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Agilent Technologies

Agilent 1260 Infinity Fluorescence Detector

User Manual

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Edition11/2013

Printed in Germany

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Safety Notices

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Agilent 1260 FLD User Manual

In This Guide

In This Guide

This manual covers

• the Agilent 1260 Infinity Fluorescence Detector (G1321B SPECTRA),

• the Agilent 1260 Infinity Fluorescence Detector (G1321C) and

• the Agilent 1200 Series Fluorescence Detector (G1321A) (obsolete).

1 Introduction to the Fluorescence Detector

This chapter gives an introduction to the detector and instrument overview.

2 Site Requirements and Specifications

This chapter provides information on environmental requirements, physical and performance specifications.

3 Installing the Module

This chapter gives information about the preferred stack setup for your system and the installation of the module.

4 Using the Fluorescence Detector

This chapter guides you how to start the work with the detector.

5 Optimizing the Detector

This chapter provides information on how to optimize the detector.

6 Troubleshooting and Diagnostics

This chapter gives an overview about the troubleshooting and diagnostic features and the different user interfaces.

Agilent 1260 FLD User Manual 3

In This Guide

7 Error Information

This chapter describes the meaning of error messages, and provides information on probable causes and suggested actions how to recover from error conditions.

8 Test Functions

This chapter describes the detector’s built in test functions.

9 Maintenance

This chapter provides general information on maintenance of the detector.

10 Parts for Maintenance

This chapter provides information on parts for maintenance.

11 Identifying Cables

This chapter provides information on cables used with the Agilent 1200 Infinity Series modules.

12 Hardware Information

This chapter describes the detector in more detail on hardware and electronics.

13 Appendix

This chapter provides safetey and other general information.

4 Agilent 1260 FLD User Manual

Contents

Contents

1 Introduction to the Fluorescence Detector 9

Introduction to the Detector 10How the Detector Operates 12Raman Effect 15Optical Unit 16Analytical Information From Primary Data 24System Overview 29Bio-inert Materials 32

2 Site Requirements and Specifications 35

Site Requirements 36Physical Specifications 39Performance Specifications 40

3 Installing the Module 49

Unpacking the Module 50Optimizing the Stack Configuration 52Installation Information on Leak and Waste Handling 57Installing the Module 61Flow Connections to the Module 64Installing Capillaries 68

4 Using the Fluorescence Detector 75

Leak and Waste Handling 76Before You Start 77Getting Started and Checkout 78Method Development 82Example: Optimization for Multiple Compounds 99How to collect spectra with modes SPECTRA ALL IN PEAK and APEX SPECTRA ONLY 109Solvent Information 113


Contents

5 Optimizing the Detector 119

Optimization Overview 120Design Features Help Optimization 122Finding the Best Wavelengths 123Finding the Best Signal Amplification 125Changing the Xenon Flash Lamp Frequency 131Selecting the Best Response Time 133Reducing Stray Light 135

6 Troubleshooting and Diagnostics 137

Overview of the Module’s Indicators and Test Functions 138Status Indicators 139User Interfaces 141Agilent Lab Advisor Software 142

7 Error Information 143

What Are Error Messages 145General Error Messages 146Detector Error Messages 151

8 Test Functions 157

Introduction 158Diagram of Light Path 159Lamp Intensity Test 160Raman ASTM Signal-to-Noise Test 162Using the Built-in Test Chromatogram 166Wavelength Verification and Calibration 168Wavelength Accuracy Test 171Wavelength Calibration Procedure 177


Contents

9 Maintenance 183

Introduction to Maintenance 184Warnings and Cautions 185Overview of Maintenance 187Cleaning the Module 188Exchanging a Flow Cell 189How to use the Cuvette 193Flow Cell Flushing 194Correcting Leaks 195Replacing Leak Handling System Parts 196Replacing the Interface Board 197Replacing Module Firmware 198Tests and Calibrations 199

10 Parts for Maintenance 201

Overview of Maintenance Parts 202Cuvette Kit 203Accessory Kit 204

11 Identifying Cables 207

Cable Overview 208Analog Cables 210Remote Cables 212BCD Cables 215CAN/LAN Cables 217External Contact Cable 218Agilent Module to PC 219

12 Hardware Information 221

Firmware Description 222Optional Interface Boards 225Electrical Connections 229Interfaces 232Setting the 8-bit Configuration Switch (without On-board LAN) 239Early Maintenance Feedback 243Instrument Layout 244


Contents

13 Appendix 245

General Safety Information 246The Waste Electrical and Electronic Equipment (WEEE) Directive (2002/96/EC) 249Lithium Batteries Information 250Radio Interference 251Sound Emission 252UV-Radiation (UV-lamps only) 253Solvent Information 254Installation of Stainless Steel Cladded PEEK Capillaries 256Agilent Technologies on Internet 262



1Introduction to the Fluorescence Detector

Introduction to the Detector 10

How the Detector Operates 12

Raman Effect 15

Optical Unit 16

Reference System 23

Analytical Information From Primary Data 24

Fluorescence Detection 24

Phosphorescence Detection 25

Processing of Raw Data 25

System Overview 29

Leak and Waste Handling 29

Bio-inert Materials 32

This chapter gives an introduction to the detector and instrument overview.

9Agilent Technologies

1 Introduction to the Fluorescence DetectorIntroduction to the Detector

Introduction to the Detector

Detector Versions

The detector is designed for highest optical performance, GLP compliance and easy maintenance. It includes the following features:

• flash lamp for highest intensity and lowest detection limit

• multi- wavelength mode for online spectra (G1321B SPECTRA)

• spectra acquisition and simultaneous multi- signal detection (G1321B SPECTRA)

• optional cuvette is available and can be used for offline measurements

• easy front access to flow cell for fast replacement and

• built- in wavelength accuracy verification.

For specifications, see “Performance Specifications” on page 40

Table 1 Detector versions

Version Description

G1321C Introduced as 1260 Infinity FLD without spectra and multi-signal capabilities in June 2013. Maximum data rate is 74 Hz Instrument firmware is A.06.54. Controlled by Instant Pilot with firmware B.02.16, Driver A.02.08, Agilent OpenLAB CDS ChemStation Edition C.01.05, OpenLAB EZChromEdition EE A.04.05, ICF A.02.01 and Lab Advisor B.02.04. The G1321C cannot be converted to G1321A/B.

G1321B SPECTRA Introduced as 1260 Infinity FLD with spectra and multi-signal capabilities in June 2010. Maximum data rate is 74 Hz. The G1321B can be converted to G1321A (emulation mode). With the introduction of the G1321C the data rate was increased to maximum 144.9 Hz (instrument firmware A.06.54).

G1321A Introduced as 1100 Series FLD with spectra and multi-signal capabilities in August 1998. Maximum data rate is 18 Hz. Obsoleted with introduction of the G1321B FLD.


Introduction to the Fluorescence Detector 1Introduction to the Detector

Figure 1 The Agilent 1260 Infinity Fluorescence Detector


1 Introduction to the Fluorescence DetectorHow the Detector Operates

How the Detector Operates

Luminescence Detection

Luminescence, the emission of light, occurs when molecules change from an excited state to their ground state. Molecules can be excited by different forms of energy, each with its own excitation process. For example, when the excitation energy is light, the process is called photoluminescence.

In basic cases, the emission of light is the reverse of absorption, see Figure 2 on page 12. With sodium vapor, for example, the absorption and emission spectra are a single line at the same wavelength. The absorption and emission spectra of organic molecules in solution produce bands instead of lines.

Figure 2 Absorption of Light Versus Emission of Light

When a more complex molecule transforms from its ground energy state into an excited state, the absorbed energy is distributed into various vibrational and rotational sub- levels. When this, same molecule returns to the ground state, this vibrational and rotational energy is first lost by relaxation without any radiation. Then the molecule transforms from this

ν

ν


Introduction to the Fluorescence Detector 1How the Detector Operates

energy level to one of the vibrational and rotational sub- levels of its ground state, emitting light, see Figure 3 on page 13. The characteristic maxima of absorption for a substance is its λEX, and for emission its λEM.

Figure 3 Relationship of Excitation and Emission Wavelengths

Photoluminescence is the collective name for two phenomena, fluorescence and phosphorescence, which differ from each other in one characteristic

way- - the delay of emission after excitation. If a molecule emits light 10- 9

to 10- 5 seconds after it was illuminated then the process was

fluorescence. If a molecule emits light longer than 10- 3 seconds after illumination then the process was phosphorescence.

Phosphorescence is a longer process because one of the electrons involved in the excitation changes its spin, during a collision with a molecule of solvent, for example. The excited molecule is now in a so- called triplet state, T, see Figure 4 on page 14.

λ


1 Introduction to the Fluorescence DetectorHow the Detector Operates

Figure 4 Phosphorescence Energy Transitions

The molecule must change its spin back again before it can return to its ground state. Since the chance of colliding with another molecule with the necessary spin for change is slight, the molecule remains in its triplet state for some time. During the second spin change the molecule loses more energy by relaxing without radiation. The light which is emitted during phosphorescence therefore has less energy and is at a longer wavelength than fluorescence.

Formula:

where

E Energy

h Planck's constant

λ Wavelength

c speed of light

E =lhc


Introduction to the Fluorescence Detector 1Raman Effect

Raman Effect

The Raman effect arises when the incident light excites molecules in the sample which subsequently scatter the light. While most of this scattered light is at the same wavelength as the incident light, some is scattered at a different wavelength. This inelastically scattered light is called Raman scatter. It results from the molecule changing it's molecular motions.

Figure 5 Raman

The energy difference between the incident light (Ei) and the Raman scattered light (Es) is equal to the energy involved in changing the molecule's vibrational state (i.e. getting the molecule to vibrate, Ev). This energy difference is called the Raman shift.

Ev = Ei - Es

Several different Raman shifted signals will often be observed; each being associated with different vibrational or rotational motions of molecules in the sample. The particular molecule and its environment will determine what Raman signals will be observed (if any).

A plot of Raman intensity versus Raman shift is a Raman spectrum.


1 Introduction to the Fluorescence DetectorOptical Unit

Optical Unit

All the elements of the optical system, shown in Figure 6 on page 17, including Xenon flash lamp, excitation condenser lens, excitation slit, mirror, excitation grating, flow cell, emission condenser lens, cut- off filter, emission slit, emission grating and photomultiplier tube are housed in the metal casting inside the detector compartment. The fluorescence detector has grating/grating optics, enabling the selection of both excitation and emission wavelengths. The flow cell can be accessed from the front of the fluorescence detector.


Introduction to the Fluorescence Detector 1Optical Unit

Figure 6 Optical Unit

The radiation source is a xenon flash- lamp. The 3 µs flash produces a continuous spectrum of light from 200 nm to 900 nm. The light output distribution can be expressed as a percentage in 100 nm intervals, see Figure 7 on page 18. The lamp can be used for some 1000 hours depending on the sensitivity requirements. You can economize during automatic operation using keyboard setpoints, so the lamp flashes during your analysis only. The lamp can be used until it no longer ignites, but the noise level may increase with usage.



UV degradation, especially below 250 nm is significantly higher compared to Visible wavelength range. Generally the "LAMP ON during run" - setting or using "economy mode" will increase lamp life by a magnitude.

Figure 7 Lamp Energy Distribution (vendor data)

The radiation emitted by the lamp is dispersed and reflected by the excitation monochromator grating onto the cell entrance slit.

The holographic concave grating is the main part of the monochromator, dispersing and reflecting the incident light. The surface contains many minute grooves, 1200 of them per millimeter. The grating carries a blaze to show improved performance in the visible range.



Figure 8 Mirror Assembly

The geometry of the grooves is optimized to reflect almost all of the

incident light, in the 1st order and disperse it with about 70 % efficiency in the ultraviolet range. Most of the remaining 30 % of the light is reflected at zero order, with no dispersion. Figure 9 on page 20 illustrates the light path at the surface of the grating.



Figure 9 Dispersion of Light by a Grating

The grating is turned using a 3- phase brushless DC motor, the position of the grating determining the wavelength or wavelength range of the light falling onto the flow cell. The grating can be programmed to change its position and therefore the wavelength during a run.

For spectra acquisition and multi- wavelength detection, the grating rotates at 4000 rpm.

The excitation and emission gratings are similar in design, but have

different blaze wavelengths. The excitation grating reflects most 1st order light in the ultraviolet range around 250 nm, whereas the emission grating reflects better in the visible range around 400 nm.

The flow cell is a solid fused silica body with a maximum back pressure of 20 bar. Excessive back pressure will result in destruction of the cell.



Operating the detector close to waste with low back pressure is recommended. A slit is integrated to the body.

Figure 10 Cross-Section of Flow Cell

The luminescence from the sample in the flow cell is collected at right angles to the incident light by a second lens, and passes through a second slit. Before the luminescence reaches the emission monochromator, a cut- off filter removes light below a certain wavelength, to reduce noise

from 1st order scatter and 2nd order stray light, see Figure 9 on page 20.

The selected wavelength of light is reflected onto the slit in the wall of the photomultiplier compartment of the optical unit. The bandwidth of the emitted light is 20 nm.



On the photocathode, Figure 11 on page 22, incident photons generate electrons. These electrons are accelerated by an electrical field between several arc- shaped dynodes. Depending on the voltage difference between any pair of dynodes, an incident electron may spark- off further electrons which accelerate onto the next dynode. An avalanche effect results: finally so many electrons are generated that a current can be measured. The amplification is a function of the voltage at the dynodes and is microprocessor controlled. You can set the amplification using the PMTGAIN function.

Figure 11 Photo-multiplier Tube

This type of so- called side- on photomultiplier is compact ensuring fast response, conserving the advantages of the short optical path shown in Figure 6 on page 17.

PMTs are designed for specific wavelength ranges. The standard PMT offers optimum sensitivity from 200 to 600 nm. In the higher wavelength range a red- sensitive PMT can improve performance.



Reference System

A reference diode, located behind the flow cell, measures the excitation (EX) light transmitted by the flow cell and corrects flash lamp fluctuations and long- term intensity drift. Because of a non- linear output of the diode (depending on the EX- wavelength), the measured data are normalized.

A diffuser is located in front of the reference diode (see Figure 6 on page 17). This diffuser is made of quartz, reduces light and allows integral measurement of the light.


1 Introduction to the Fluorescence DetectorAnalytical Information From Primary Data

Analytical Information From Primary Data

We now know how the primary data from your sample is acquired in the optical unit. But how can the data be used as information in analytical chemistry? Depending on the chemistry of your application, the luminescence measured by the fluorescence detector will have different characteristics. You must decide, using your knowledge of the sample, what mode of detection you will use.

Fluorescence Detection

When the lamp flashes, the fluorescing compounds in your sample will luminesce almost simultaneously, see Figure 12 on page 24. The luminescence is short- lived, therefore the fluorescence detector need only measure over a short period of time after the lamp has flashed.

Figure 12 Measurement of Fluorescence


Introduction to the Fluorescence Detector 1Analytical Information From Primary Data

Phosphorescence Detection

An appropriate parameter set will be specified as soon as you chose the phosphorescence detection mode (special setpoints under FLD parameter settings).

Figure 13 Measurement of Phosphorescence

Processing of Raw Data

If the lamp flashes at single wavelength and high- power, then the fluorescence data rate is 296 Hz. That means that your sample is illuminated 296 times per second, and any luminescence generated by the components eluted from the column is measured 296 times per second.

If the “economy” or multi- wavelength mode is set, then the flash frequency is 74 Hz.



Figure 14 LAMP: Frequency of Flash, Fluorescence, and Phosphorescence

You can improve the signal- to- noise characteristics by disabling the “economy” mode.

The data resolution is 20 bit at a response time of 4 s (default, which is equivalent to a time constant of 1.8 s and appropriate for standard chromatographical conditions). Weak signals may cause errors in quantification because of insufficient resolution. Check your proposed PMTGAIN. If it is significantly distant from your setting, change your method or check the purity of your solvent. See also “Finding the Best Signal Amplification” on page 125.

You can amplify the signal using PMTGAIN. Depending on the PMTGAIN you have set, a multiple of electrons is generated for every photon falling on the photomultiplier. You can quantify large and small peaks in the same chromatogram by adding PMTGAIN changes during the run into a timetable.

NOTE Disabling the “economy” mode will shorten the lifetime of the lamp significantly. Consider lifetime saving by switching off the lamp after the run is completed.


Introduction to the Fluorescence Detector 1Analytical Information From Primary Data

Figure 15 PMTGAIN: Amplification of Signal

Check proposed PMTGAIN. Deviations of more than 2 PMT gains should be corrected in the method.

Each PMTGAIN step is increased approximately by a factor of 2 (range 0 - 18). To optimize your amplification for the peak with the highest emission, raise the PMTGAIN setting until the best signal- to- noise is achieved.

After the photons are converted and multiplied into an electronic signal, the signal (at present analog) is tracked and held beyond the photomultiplier. After being held, the signal is converted by an A- to- D converter to give one raw data point (digital). Eleven of these data points are bunched together as the first step of data processing. Bunching improves your signal- to- noise ratio.

The bunched data, shown as larger black dots in Figure 16 on page 28, is then filtered using a boxcar filter. The data is smoothed, without being reduced, by taking the mean of a number of points. The mean of the same points minus the first plus the next, and so on, is calculated so that there are the same number of bunched and filtered points as the original bunched points. You can define the length of the boxcar element using the RESPONSETIME function: the longer the RESPONSETIME, the greater the number of data points averaged. A four- fold increase in RESPONSETIME (for example, 1 sec to 4 sec) doubles the signal- to- noise ratio.



Figure 16 RESPONSETIME: Signal-to-Noise Ratio


Introduction to the Fluorescence Detector 1System Overview

System Overview

Leak and Waste Handling

The 1200 Infinity Series has been designed for safe leak and waste handling. It is important that all security concepts are understood and instructions are carefully followed.


1 Introduction to the Fluorescence DetectorSystem Overview

Figure 17 Leak and waste handling concept (overview - typical stack configuration as an example)


Introduction to the Fluorescence Detector 1System Overview

The solvent cabinet (1) is designed to store a maximum volume of 6 L solvent. The maximum volume for an individual bottle stored in the solvent cabinet should not exceed 2.5 L. For details, see the usage guideline for the Agilent 1200 Infinity Series Solvent Cabinets (a printed copy of the guideline has been shipped with the solvent cabinet, electronic copies are available on the Internet).

The leak pan (2) (individually designed in each module) guides solvents to the front of the module. The concept covers also leakages on internal parts (e.g. the detector’s flow cell). The leak sensor in the leak pan stops the running system as soon as the leak detection level is reached.

The leak pan's outlet port (3, A) guides excessive overfill from one module to the next, as the solvent flows into the next module’s leak funnel (3, B) and the connected corrugated waste tube (3, C). The corrugated waste tube guides the solvent to the next lower positioned module’s leak tray and sensor.

The waste tube of the sampler’s needle wash port (4) guides solvents to waste.

The condense drain outlet of the autosampler cooler (5) guides condensate to waste.

The waste tube of the purge valve (6) guides solvents to waste.

The waste tube connected to the leak pan outlet on each of the bottom instruments (7) guides the solvent to a suitable waste container.


1 Introduction to the Fluorescence DetectorBio-inert Materials

Bio-inert Materials

For the Agilent 1260 Infinity Bio- inert LC system, Agilent Technologies uses highest quality materials in the flow path (also referred to as wetted parts), which are widely accepted by life scientists, as they are known for optimum inertness to biological samples and ensure best compatibility with common samples and solvents over a wide pH range. Explicitly, the complete flow path is free of stainless steel and free of other alloys containing metals such as iron, nickel, cobalt, chromium, molybdenum or copper, which can interfere with biological samples. The flow downstream of the sample introduction contains no metals whatsoever.

Table 2 Bio-inert materials used in Agilent 1260 Infinity Systems

Module Materials

Agilent 1260 Infinity Bio-inert Quaternary Pump (G5611A)

Titanium, gold, platinum-iridium, ceramic, ruby, PTFE, PEEK

Agilent 1260 Infinity Bio-inert High-Performance Autosampler(G5667A)

Upstream of sample introduction:• Titanium, gold, PTFE, PEEK, ceramic

Downstream of sample introduction:• PEEK, ceramic

Agilent 1260 Infinity Bio-inert Manual Injector(G5628A)

PEEK, ceramic

Agilent 1260 Infinity Bio-inert Analytical Fraction Collector(G5664A)

PEEK, ceramic, PTFE

Bio-inert Flow Cells:

Standard flow cell bio-inert, 10 mm, 13 µL, 120 bar ( 12 MPa) for MWD/DAD, includes Capillary Kit Flow Cells BIO (p/n G5615-68755) (G5615-60022) (for Agilent 1260 Infinity Diode Array Detectors DAD G1315C/D)

PEEK, ceramic, sapphire, PTFE

Max-Light Cartridge Cell Bio-inert ( 10 mm, V(s) 1.0 µL) (G5615-60018) andMax-Light Cartridge Cell Bio-inert ( 60 mm, V(s) 4.0 µL) (G5615-60017) (for Agilent 1200 Infinity Series Diode Array Detectors DAD G4212A/B)

PEEK, fused silica

Bio-inert flow cell, 8 µL, 20 bar (pH 1–12) includes Capillary Kit Flow Cells BIO (p/n G5615-68755) (G5615-60005) (for Agilent 1260 Infinity Fluorescence Detector FLD G1321B)

PEEK, fused silica, PTFE


Introduction to the Fluorescence Detector 1Bio-inert Materials

Bio-inert heat-exchanger G5616-60050(for Agilent 1290 Infinity Thermostatted Column Compartment G1316C)

PEEK (steel-cladded)

Bio-inert Valve heads G4235A, G5631A, G5639A: PEEK, ceramic (Al2O3 based)

Bio-inert Connection capillaries Upstream of sample introduction:• Titanium

Downstream of sample introduction:• Agilent uses stainless-steel-cladded

PEEK capillaries, which keep the flow path free of steel and provide pressure stability to more than 600 bar.

Table 2 Bio-inert materials used in Agilent 1260 Infinity Systems

Module Materials

NOTE To ensure optimum bio-compatibility of your Agilent 1260 Infinity Bio-inert LC system, do not include non-inert standard modules or parts to the flow path. Do not use any parts that are not labeled as Agilent “Bio-inert”. For solvent compatibility of these materials, see “Material Information” on page 113.


1 Introduction to the Fluorescence DetectorBio-inert Materials



2Site Requirements and Specifications

Site Requirements 36

Physical Specifications 39

Performance Specifications 40

This chapter provides information on environmental requirements, physical and performance specifications.


2 Site Requirements and SpecificationsSite Requirements

Site Requirements

A suitable environment is important to ensure optimal performance of the instrument.

Power Considerations

The module power supply has wide ranging capability. It accepts any line voltage in the range described in Table 3 on page 39. Consequently there is no voltage selector in the rear of the module. There are also no externally accessible fuses, because automatic electronic fuses are implemented in the power supply.

WARNING Hazard of electrical shock or damage of your instrumentation

can result, if the devices are connected to a line voltage higher than specified.

➔ Connect your instrument to the specified line voltage only.

WARNING The module is partially energized when switched off, as long as the power cord is plugged in.

Repair work at the module can lead to personal injuries, e.g. electrical shock, when the cover is opened and the module is connected to power.

➔ Always unplug the power cable before opening the cover.

➔ Do not connect the power cable to the instrument while the covers are removed.

CAUTION Inaccessible power plug.

In case of emergency it must be possible to disconnect the instrument from the power line at any time.

➔ Make sure the power connector of the instrument can be easily reached and unplugged.

➔ Provide sufficient space behind the power socket of the instrument to unplug the cable.


Site Requirements and Specifications 2Site Requirements

Power Cords

Different power cords are offered as options with the module. The female end of all power cords is identical. It plugs into the power- input socket at the rear. The male end of each power cord is different and designed to match the wall socket of a particular country or region.

WARNING Absence of ground connection or use of unspecified power cord

The absence of ground connection or the use of unspecified power cord can lead to electric shock or short circuit.

➔ Never operate your instrumentation from a power outlet that has no ground connection.

➔ Never use a power cord other than the Agilent Technologies power cord designed for your region.

WARNING Use of unsupplied cables

Using cables not supplied by Agilent Technologies can lead to damage of the electronic components or personal injury.

➔ Never use cables other than the ones supplied by Agilent Technologies to ensure proper functionality and compliance with safety or EMC regulations.

WARNING Unintended use of supplied power cords

Using power cords for unintended purposes can lead to personal injury or damage of electronic equipment.

➔ Never use the power cords that Agilent Technologies supplies with this instrument for any other equipment.


2 Site Requirements and SpecificationsSite Requirements

Bench Space

The module dimensions and weight (see Table 3 on page 39) allow you to place the module on almost any desk or laboratory bench. It needs an additional 2.5 cm (1.0 inches) of space on either side and approximately 8 cm (3.1 inches) in the rear for air circulation and electric connections.

If the bench shall carry a complete HPLC system, make sure that the bench is designed to bear the weight of all modules.

The module should be operated in a horizontal position.

Condensation

CAUTION Condensation within the module

Condensation will damage the system electronics.

➔ Do not store, ship or use your module under conditions where temperature fluctuations could cause condensation within the module.

➔ If your module was shipped in cold weather, leave it in its box and allow it to warm slowly to room temperature to avoid condensation.


Site Requirements and Specifications 2Physical Specifications

Physical Specifications

Table 3 Physical Specifications

Type Specification Comments

Weight 11.5 kg (26 lbs)

Dimensions (height × width × depth)

140 x 345 × 435 mm (7 x 13.5 × 17 inches)

Line voltage 100 – 240 VAC, ± 10 % Wide-ranging capability

Line frequency 50 or 60 Hz, ± 5 %

Power consumption 180 VA / 70 W / 239 BTU Maximum

Ambient operating temperature

0 - 40 °C (32 - 104 °F)

Ambient non-operating temperature

-40 – 70 °C (-40 – 158 °F)

Humidity < 95 % r.h. at 40 °C (104 °F) Non-condensing

Operating altitude Up to 2000 m (6562 ft)

Non-operating altitude Up to 4600 m (15091 ft) For storing the module

Safety standards: IEC, CSA, UL

Installation category II, Pollution degree 2 For indoor use only.


2 Site Requirements and SpecificationsPerformance Specifications

Performance Specifications

Table 4 Performance Specifications Agilent 1260 Infinity Fluorescence Detector (G1321B)


Detection type Multi-signal fluorescence detector with rapid on-line scanning capabilities and spectral data analysis

Performance specifications Single wavelength operation:• RAMAN (H2O) > 500 (noise reference

measured at signal)

Ex=350 nm, Em=397 nm, dark value 450 nm, standard flow cell

• RAMAN (H2O) > 3000 (noise

reference measured at dark value)


Dual wavelength operation:RAMAN (H2O) > 300 Ex 350 nm, Em

397 nm and Ex 350 nm, Em 450 nm, standard flow cell.

see note below this tablesee Service Manual for details

Light source Xenon Flash Lamp, normal mode 20 W, economy mode 5 W, lifetime 4000 h

Pulse frequency 296 Hz for single signal mode74 Hz for economy mode

Maximum data rate 74 Hz, 145 Hz 145 Hz with firmware A.06.54 and above

Excitation monochromator Range: settable 200 nm - 1200 nm and zero-orderBandwidth: 20 nm (fixed)Monochromator: concave holographic grating, F/1.6, blaze: 300 nm


Site Requirements and Specifications 2Performance Specifications

Emission monochromator Range: settable 200 nm - 1200 nm and zero-orderBandwidth: 20 nm (fixed)Monochromator: concave holographic grating, F/1.6, blaze: 400 nm

Reference system in-line excitation measurement

Timetable programing up to 4 signal wavelengths, response time, PMT Gain, baseline behavior (append, free, zero), spectral parameters

Spectrum acquisition Excitation or Emission spectraScan speed: 28 ms per datapoint (e.g. 0.6 s/spectrum 200 – 400 nm, 10 nm step)Step size: 1 – 20 nmSpectra storage: All

Wavelength characteristic Repeatability +/- 0.2 nmAccuracy +/- 3 nm setting

Flow cells Standard: 8 µL volume and 20 bar (2 MPa) pressure maximum, fused silica block

Optional:• Fluorescence cuvette for offline

spectroscopic measurements with 1 mL syringe, 8 µL volume

• Bio-inert: 8 µL volume and 20 bar (2 MPa) pressure maximum, (pH 1–12)

• Micro: 4 µL volume and 20 bar (2 MPa) pressure maximum

Control and data evaluation Agilent ChemStation for LC, Agilent Instant Pilot G4208A with limited spectral data analysis and printing of spectra





Analog outputs Recorder/integrator: 100 mV or 1 V, output range > 100 LU, two outputs

100 LU is the recommended range, see "FLD Scaling Range and Operating Conditions"

Communications Controller-area network (CAN), RS-232C, LAN, APG Remote: ready, start, stop and shut-down signals

Safety and maintenance Extensive support for troubleshooting and maintenance is provided by the Instant Pilot, Agilent Lab Advisor, and the Chromatography Data System. Safety-related features are leak detection, safe leak handling, leak output signal for shutdown of pumping system, and low voltages in major maintenance areas.

GLP features Early maintenance feedback (EMF) for continuous tracking of instrument usage in terms of lamp burn time with user-settable limits and feedback messages. Electronic records of maintenance and errors. Verification of wavelength accuracy, using the Raman band of water.

Housing All materials recyclable.

Environment 0 – 40 °C constant temperature at <95 % humidity (non-condensing)

Dimensions 140 mm x 345 mm x 435 mm (5.5 x 13.5 x 17 inches) (height x width x depth)

Weight 11.5 kg (25.5 lbs)





Table 5 Performance Specifications Agilent 1260 Infinity Fluorescence Detector (G1321C)


Detection type One signal wavelength (excitation and emission)

Programmable single wavelength (excitation and emission) fluorescence detector


measured at signal)


• RAMAN (H2O) > 3000 (noise

reference measured at dark value)





Maximum data rate 74 Hz

















Safety and maintenance Extensive support for troubleshooting and maintenance is provided by the Instant Pilot, Agilent Lab Advisor, and the Chromatography Data System. Safety-related features are leak detection, safe leak handling, leak output signal for shutdown of pumping system, and low voltages in major maintenance areas.





Table 6 Performance Specifications Agilent 1200 Series Fluorescence Detector (G1321A)


Detection type Multi-signal fluorescence detector with rapid on-line scanning capabilities and spectral data analysis


measured at signal)


Dual wavelength operation:RAMAN (H2O) > 300 Ex 350 nm, Em

397 nm and Ex 350 nm, Em 450 nm, standard flow cell.




Maximum data rate 37 Hz







Spectrum acquisition Excitation or Emission spectraScan speed: 28 ms per datapoint (e.g. 0.6 s/spectrum 200 – 400 nm, 10 nm step)Step size: 1 – 20 nmSpectra storage: All















Safety and maintenance Extensive diagnostics, error detection and display (through Instant Pilot G4208A and ChemStation), leak detection, safe leak handling, leak output signal for shutdown of pumping system. Low voltages in major maintenance areas.










3Installing the Module

Unpacking the Module 50

Optimizing the Stack Configuration 52

One Stack Configuration 53

Two Stack Configuration 55

Installation Information on Leak and Waste Handling 57

Installing the Module 61

Flow Connections to the Module 64

Installing Capillaries 68

This chapter gives information about the preferred stack setup for your system and the installation of the module.


3 Installing the ModuleUnpacking the Module

Unpacking the Module

Damaged Packaging

If the delivery packaging shows signs of external damage, please call your Agilent Technologies sales and service office immediately. Inform your service representative that the instrument may have been damaged during shipment.

CAUTION "Defective on arrival" problems

If there are signs of damage, please do not attempt to install the module. Inspection by Agilent is required to evaluate if the instrument is in good condition or damaged.

➔ Notify your Agilent sales and service office about the damage.

➔ An Agilent service representative will inspect the instrument at your site and initiate appropriate actions.


Installing the Module 3Unpacking the Module

Delivery Checklist

Ensure all parts and materials have been delivered with your module. The delivery checklist is shown below.

For parts identification please check the illustrated parts breakdown in “Parts for Maintenance” on page 201

Please report any missing or damaged parts to your local Agilent Technologies sales and service office.

Table 7 Detector Checklist

Description Quantity

Detector 1

Power cable 1

CAN cable 1

Flow cell as ordered

Optional flow cell/cuvette as ordered

User Manual on Documentation CD (part of the shipment - not module specific)

1 per order

Accessory kit (see “Standard Accessory Kit” on page 204)

1


3 Installing the ModuleOptimizing the Stack Configuration

Optimizing the Stack Configuration

If your module is part of a complete Agilent 1260 Infinity Liquid Chromatograph, you can ensure optimum performance by installing the following configurations. These configurations optimize the system flow path, ensuring minimum delay volume.


Installing the Module 3Optimizing the Stack Configuration

One Stack Configuration

Ensure optimum performance by installing the modules of the Agilent 1260 Infinity LC System in the following configuration (See Figure 18 on page 53 and Figure 19 on page 54). This configuration optimizes the flow path for minimum delay volume and minimizes the bench space required.

Figure 18 Recommended Stack Configuration for 1260 Infinity (Front View)



Figure 19 Recommended Stack Configuration for 1260 Infinity (Rear View)


Installing the Module 3Optimizing the Stack Configuration

Two Stack Configuration

To avoid excessive height of the stack when the autosampler thermostat is added to the system it is recommended to form two stacks. Some users prefer the lower height of this arrangement even without the autosampler thermostat. A slightly longer capillary is required between the pump and autosampler. (See Figure 20 on page 55 and Figure 21 on page 56).

Figure 20 Recommended Two Stack Configuration for 1260 Infinity (Front View)



Figure 21 Recommended Two Stack Configuration for 1260 Infinity (Rear View)


Installing the Module 3Installation Information on Leak and Waste Handling

Installation Information on Leak and Waste Handling

The Agilent 1200 Infinity Series has been designed for safe leak and waste handling. It is important that all security concepts are understood and instructions are carefully followed.

WARNING Toxic, flammable and hazardous solvents, samples and reagents

The handling of solvents, samples and reagents can hold health and safety risks.

➔ When working with these substances observe appropriate safety procedures (for example by wearing goggles, safety gloves and protective clothing) as described in the material handling and safety data sheet supplied by the vendor, and follow good laboratory practice.

➔ The volume of substances should be reduced to the minimum required for the analysis.

➔ Never exceed the maximal permissible volume of solvents (6 L) in the solvent cabinet.

➔ Do not use bottles that exceed the maximum permissible volume as specified in the usage guideline for the Agilent 1200 Infinity Series Solvent Cabinets.

➔ Arrange the bottles as specified in the usage guideline for the solvent cabinet.

➔ A printed copy of the guideline has been shipped with the solvent cabinet, electronic copies are available on the Internet.

NOTE Recommendations for Solvent Cabinet

For details, see the usage guideline for the Agilent 1200 Infinity Series Solvent Cabinets.


3 Installing the ModuleInstallation Information on Leak and Waste Handling

Figure 22 Leak and waste handling (overview - typical stack configuration as an example)


Installing the Module 3Installation Information on Leak and Waste Handling

1 Stack the modules according to the adequate stack configuration.

The leak pan outlet of the upper module must be vertically positioned above the leak tray of the lower module, see Figure 22 on page 58.

2 Connect data and power cables to the modules, see section Installing the Module below.

3 Connect capillaries and tubes to the modules, see section Flow Connections to the module below or the relevant system manual.

1 Solvent cabinet

2 Leak pan

3 Leak pan's outlet port (A), leak funnel (B) and corrugated waste tube (C)

4 Waste tube of the sampler’s needle wash

5 Condense drain outlet of the autosampler cooler

6 Waste tube of the purge valve

7 Waste tube


➔ Keep solvent path free from blockages.

➔ Keep the flow path closed (in case the pump in the system is equipped with a passive inlet valve, solvent may leak out due to hydrostatic pressure, even if your instrument is off).

➔ Avoid loops.

➔ Tubes must not sag.

➔ Do not bend tubes.

➔ Do not immerse tube end in waste liquid.

➔ Do not intubate tubes in other tubes.

➔ For correct tubing follow instructions on label attached to the module.


3 Installing the ModuleInstallation Information on Leak and Waste Handling

Figure 23 Warning label (illustration for correct waste tubing)


Installing the Module 3Installing the Module

Installing the Module

For other cables see “Cable Overview” on page 208.

1 Install the LAN interface board in the detector (if required), see “Replacing the Interface Board” on page 197.

2 Place the detector in the stack or on the bench in a horizontal position.

Parts required Description

Power cord

Software required Agilent Data System and/or Instant Pilot G4208A.

Preparations Locate bench space

Provide power connections

Unpack the detector

WARNING Module is partially energized when switched off, as long as the power cord is plugged in.

Repair work at the module can lead to personal injuries, e.g. shock hazard, when the cover is opened and the module is connected to power.

➔ Make sure that it is always possible to access the power plug.

➔ Remove the power cable from the instrument before opening the cover.

➔ Do not connect the power cable to the Instrument while the covers are removed.


3 Installing the ModuleInstalling the Module

3 Ensure the line power switch at the front of the detector is OFF.

Figure 24 Front View of Detector

4 Connect the power cable to the power connector at the rear of the detector.

5 Connect the CAN cable to other modules.

6 If an Agilent ChemStation is the controller, connect the LAN connection to the LAN interface board in the detector.

7 Connect the analog cable(s) (optional).

8 Connect the APG remote cable (optional) for non- Agilent Series instruments.

NOTE The detector (DAD/MWD/FLD/VWD/RID) is the preferred access point for control via LAN (due to higher data load).


Installing the Module 3Installing the Module

9 Turn ON power by pushing the button at the lower left hand side of the detector. The status LED should be green.

Figure 25 Rear View of Detector

NOTE The detector is turned ON when the line power switch is pressed and the green indicator lamp is illuminated. The detector is turned OFF when the line power switch is protruding and the green light is OFF.

NOTE The detector was shipped with default configuration settings.

NOTE The GPIB interface has been removed with the introduction of the 1260 Infinity modules.


3 Installing the ModuleFlow Connections to the Module

Flow Connections to the Module

For bio-inert modules use bio-inert parts only!

Tools required Description

Wrench, 1/4 – 5/16 inch(for capillary connections)

Parts required p/n Description

G1321-68755 Accessory kit

Preparations Detector is installed in the LC system.





➔ Do not operate the instrument in an explosive atmosphere.

NOTE The flow cell is shipped with a filling of isopropanol (also recommended when the instrument and/or flow cell is shipped to another location). This is to avoid breakage due to subambient conditions.


Installing the Module 3Flow Connections to the Module

1 Press the release buttons and remove the front cover to gain access to the flow cell area.

2 Locate the flow cell.


3 Installing the ModuleFlow Connections to the Module

3 Assemble the column detector capillary from the accessory kit. One side is already factory-assembled.

4 Assemble the waste tubing from the accessory kit.

NOTEThe fluorescence detector should be the last module in the flow system. An additional detector should be installed before the fluorescence detector to prevent any overpressure to the cell (maximum 20 bar).

When working with detector behind the FLD (on own risk) determine the backpressure of this detector first by

- removing the column and the last detect and measuring system pressure at the application flow rate.

- connecting the last detector (without column and FLD) and measuring the system pressure with flow.

- the difference in measured pressure is due to the back pressure generated by the last detector and is the pressure seen by the FLD.


Installing the Module 3Flow Connections to the Module

The installation of the detector is now complete.

5 Insert the flow cell and install the capillaries to the flow cell (top is outlet, bottom is inlet).

6 Connect the waste tubing to the bottom waste fitting.

7 Establish flow and observe if leaks occur. 8 Replace the front cover.

NOTE The detector should be operated with the front cover in place to protect the flow cell area against strong drafts from the ouside.


3 Installing the ModuleInstalling Capillaries

Installing Capillaries

In May 2013, Agilent has introduced new UHP- FF fittings, which are designed for improved robustness and ease of use. Previous fittings require careful handling. Therefore it is important to know, which fittings are used in the system.

The figure below illustrates the differences between new and previous capillaries.

Figure 26 New bio-inert capillary and UHP-FF fitting with nose

Figure 27 Previous bio-inert capillary and fitting

NOTE For handling instructions of capillaries and fittings, used in modules before delivery of the new UHP-FF fittings (introduced in May 2013), refer to “Installation of Stainless Steel Cladded PEEK Capillaries” on page 256.

NOTE To work on bio-inert capillaries produced before May 2013, you will need a 1 /4 inch wrench instead of the 5 mm mounting tool.


Installing the Module 3Installing Capillaries

Installing UHP-FF Fittings

1 Slide the fitting on the capillary. Let the capillary jut out 5 mm.

Tools required p/n Description

5043-0915 Fitting mounting toolfor bio-inert capillaries


Capillaries and Fittings

For details refer to the part section of the manual.



2 Insert the fitting to the receiving port and push the capillary to the bottom of the port.

3 Finger tighten the nut into the port until snug.



4 Use Fitting mounting tool (5043- 0915) or a 5 mm hex wrench for fixing the fitting (maximum torque 0.8 Nm).

CAUTION Potential damage of capillaries

➔ Do not remove fittings from used capillaries.



5 When using UHP- FF fittings with bioinert capillaries, do not try to remove fittings from these capillaries. Bio- inert capillaries are using a PEEK front end, which may expand under pressure especially when being in contact with some organic solvents. If a fitting is moved across an expanded PEEK end, there is a risk of damaging the capillary by ripping off its end. Before re- installing such capillaries, push the ferrule towards the rear site for a small distance.

Figure 28 Capillary fitting



Installation of the Bio-inert Zero Dead Volume (ZDV) Union

The Bio- inert ZDV (p/n 5067- 4741) union has two different connectors where capillaries need to be installed in the correct sequence. Otherwise, an inset of the union may be damaged and the connection may not be tight.

CAUTION Potential leak or damage of the Bio-inert ZDV Union.

➔ To avoid leaks or a damage to the Bio-inert ZDV union, follow the procedure below in the prescribed sequence.

1 Install the capillary at the end marked with a ring/indentation.

2 Install the second capillary at the other end.



4Using the Fluorescence Detector

Leak and Waste Handling 76

Before You Start 77

Getting Started and Checkout 78

Starting Your Detector 78

Setting the Chromatographic Conditions 79

Observe the Maxima via the Isoabsorbance Plot 81

Method Development 82

Step 1: Check the LC System for Impurities 83

Step 2: Optimize Limits of Detection and Selectivity 84

Step 3: Set up Routine Methods 95

Example: Optimization for Multiple Compounds 99

How to collect spectra with modes SPECTRA ALL IN PEAK and APEX SPECTRA ONLY 109

Solvent Information 113

This chapter guides you how to start the work with the detector.


4 Using the Fluorescence DetectorLeak and Waste Handling

Leak and Waste Handling

For details on correct installation, see “Installation Information on Leak and Waste Handling” on page 57.






➔ Never exceed the maximal permissible volume of solvents (6 L) in the solvent cabinet.

➔ Do not use bottles that exceed the maximum permissible volume as specified in the usage guideline for the Agilent 1200 Infinity Series Solvent Cabinets.

➔ Arrange the bottles as specified in the usage guideline for the solvent cabinet.

➔ A printed copy of the guideline has been shipped with the solvent cabinet, electronic copies are available on the Internet.

➔ The residual free volume in the appropriate waste container must be large enough to collect the waste liquid.

➔ Check the filling level of the waste container regularly.

➔ To achieve maximal safety, check the correct installation regularly.

NOTE Recommendations for Solvent Cabinet

For details, see the usage guideline for the Agilent 1200 Infinity Series Solvent Cabinets.


Using the Fluorescence Detector 4Before You Start

Before You Start

Your normal LC grade solvents usually give good results most of the time. But experience shows that baseline noise can be higher (lower signal- to- noise ratio) when impurities are in the solvents.

Flush your solvent delivery system for at least 15 minutes before checking sensitivity. If your pump has multiple channels, you should also flush the channels not in use.

For optimal results refer to “Optimizing the Detector” on page 119.

NOTE Some features (e.g. spectrum acquisition, multi-wavelength detection) described in this chapter are not available on the G1321C FLD.


4 Using the Fluorescence DetectorGetting Started and Checkout

Getting Started and Checkout

This chapter describes the check out of the Agilent 1260 Infinity Fluorescence Detector using the Agilent isocratic checkout sample.

Starting Your Detector

1 Turn ON the detector.

2 Turn ON the lamp.

When the lamp is turned on the first time the instrument performs some internal checks and a calibration check which takes about 5 minutes.

3 You are now ready to change the settings of your detector.

When If you want to checkout the detector

Parts required # p/n Description

1 5063-6528 Start-up Kit, includes

1 LC Column and parts listed below

1 01080-68704 Agilent isocratic checkout sampleThis 0.5 mL ampoule contains 0.15 wt.% dimethylphthalate, 0.15 wt.% diethylphthalate, 0.01 wt.% biphenyl, 0.03 wt.% o-terphenyl in methanol.

1 0100-1516 Fitting male PEEK, 2/pk

1 5021-1817 Capillary ST 0.17 mm x 150 mm

Hardware required LC system with FLD


Using the Fluorescence Detector 4Getting Started and Checkout

Setting the Chromatographic Conditions

1 Set up the system with the following chromatographic conditions and wait until the baseline gets stable.

2 Set the FLD setpoints according to Figure 29 on page 80.

Table 8 Chromatographic Conditions

Mobile phases A = water = 35 %B = Acetonitrile = 65 %

Column OSD-Hypersil column, 125 mm x 4 mm i.d. with 5 µm particles

Sample Isocratic standard sample, 1:10 diluted in methanol

Flow rate 1.5 ml/min

Compressibility A (water) 46

Compressibility B (Acetonitrile) 115

Stroke A and B auto

Stop time 4 min

Injection volume 5 µl

Oven temperature (1200) 30 °C

FLD Excitations/Emission Wavelength EX = 246 nm, EM = 317 nm

FLD PMT Gain PMT = 10

FLD Response time 4 s


4 Using the Fluorescence DetectorGetting Started and Checkout

3 Start the run.

The resulting chromatograms are shown below :

Figure 30 Biphenyl Peak With Different Excitation Wavelengths

The excitation maxima are around 250 nm.

In this example additional excitation wavelenghts (B, C, D) are used. This will increase the scan time and may lower the performance.

Figure 29 FLD Parameters


Using the Fluorescence Detector 4Getting Started and Checkout

Observe the Maxima via the Isoabsorbance Plot

1 Load the data file (λEX = 246 nm, λEM = 317 nm) and open the isoabsorbance plot.

2 The maximum λEX will be found around 250 nm.

Figure 31 Isoabsorbance Plot


4 Using the Fluorescence DetectorMethod Development

Method Development

Fluorescence detectors are used in liquid chromatography when superior limits of detection and selectivity are required. Thorough method development, including spectra acquisition, is fundamental to achieve good results. This chapter describes three different steps that can be taken with the Agilent fluorescence detector. Table 9 on page 82 gives an overview of how to benefit from the operation modes during these steps.

Table 9 Steps for thorough method development

Step 1: Check system Step 2: Optimize limits of detection and selectivity

Step 3: Set up routine methods

Fluorescence scan Find impurities (for example, in solvents and reagents)

Determine simultaneously the excitation and emission spectra of a pure compound

Signal mode Perform wavelength switching

Use for lowest limits of detection

Spectral mode/multi-wavelength detection

Determine Ex/Em spectra for all separated compounds in a single run

Activate up to four wavelengths simultaneously

Collect online spectra, perform library search, determine peak purity

Deactivate wavelength switching


Using the Fluorescence Detector 4Method Development

Step 1: Check the LC System for Impurities

A critical issue in trace level fluorescence detection is to have an LC system free of fluorescent contamination. Most contaminants derive from impure solvents. Taking a fluorescence scan is a convenient way to check the quality of the solvent in a few minutes. This can be done, for example, by filling the FLD cuvette directly with the solvent for an offline measurement even before the start of a chromatographic run. The result can be displayed as an isofluorescence plot or a three- dimensional plot. Different colors reflect different intensities.

Figure 32 on page 83 shows a sample of slightly impure water which was planned for use as mobile phase. The area where fluorescence of the contaminated water sample can be seen is between the stray light areas: the first- and second- order Raleigh stray light and Raman stray light.

Since "excitation" and "emission" wavelength are the same for Raleigh stray light, the area of first- order Raleigh stray light is visible in the left upper area of the diagram. The Raman bands of water are seen below the first- order Raleigh stray light. Since the cut- off filter cuts off light below 280 nm, the second- order Raleigh stray light starts above 560 nm.

Stray light acts in the same way as impurities in that it simulates background noise. In both cases, a higher noise level and therefore a higher limit of detection are obtained. This indicates that high sensitivity measurements should be done away from wavelength settings that have a high stray light background.

A pure water sample was put into the flow cell. Spectra were recorded at 5 nm step sizes.

Figure 32 Isofluorescence plot of a mobile phase



Step 2: Optimize Limits of Detection and Selectivity

To achieve optimum limits of detection and selectivity, analysts must find out about the fluorescent properties of the compounds of interest. Excitation and emission wavelengths can be selected for optimum limits of detection and best selectivity. In general, fluorescence spectra obtained with different instruments may show significant differences depending on the hardware and software used.

The traditional approach is to extract an appropriate excitation wavelength from the UV spectrum that is similar to the fluorescence excitation spectrum (see Figure 33 on page 84) and to record the emission spectrum. Then with an optimum emission wavelength determined, the excitation spectrum is acquired.

Excitation spectrum with emission at 440 nm, emission spectrum with excitation at 250 nm of 1 µg/ml quinidine.Detector settings:Step size 5 nm, PMT 12 Response time 4 s.

Figure 33 Excitation and emission spectra of quinidine



These tasks have to be repeated for each compound using either a fluorescence spectrophotometer or stop- flow conditions in LC. Usually each compound requires a separate run. As a result, a set of excitation and emission spectrum is obtained (Figure 32 on page 83) for each compound. Since this is a tedious procedure, it is applicable only when there is a limited number of compounds of interest.

The Agilent 1200 Infinity Series LC offers three different ways to obtain complete information on a compound's fluorescence:

Procedure I - Take a fluorescence scan offline for a single compound as described above for the mobile phase. This is done preferably with a manual FLD cuvette when pure compounds are available.

Procedure II - Use two LC runs with the Agilent 1260 Infinity Fluorescence Detector to separate the compound mix under known conditions and acquire emission and excitation spectra separately.

Procedure III - Use an Agilent 1200 Infinty Series FLD /DAD combination and acquire UV/Visible spectra (equivalent to excitation spectra) with the DAD and emission spectra with the FLD- both in a single run.

Procedure I - Take a fluorescence scan

Because fluorescence spectra traditionally have not been easily available with previous LC fluorescence detectors, standard fluorescence spectrophotometers have been used in the past to acquire spectral information for unknown compounds. Unfortunately this approach limits optimization, as there are differences expected in optical design between an LC detector and a dedicated fluorescence spectrophotometer, or even between detectors. These differences can lead to variations for the optimum excitation and emission wavelengths.

The Agilent 1260 Infinity Fluorescence Detector offers a fluorescence scan that delivers all spectral information previously obtained with a standard fluorescence spectrophotometer, independent of the LC fluorescence detector. Figure 34 on page 87 shows the complete information for quinidine as obtained with the Agilent 1260 Infinity Fluorescence Detector and a manual cuvette in a single offline measurement. The optima for excitation and emission wavelengths can be extracted as coordinates of the maxima in the three dimensional plot. One of the three maxima in the center of the plot can be chosen to define the excitation wavelength. The selection depends on the additional compounds that are going to be



analyzed in the chromatographic run and the background noise that may be different upon excitation at 250 nm, 315 nm or 350 nm. The maximum of emission is observed at 440 nm.

Details for Figure 34 on page 87:

All excitation and emission spectra of Quinidine (1 µg/ml) are shown in graphic. Fluorescence intensity is plotted vs excitation and emission wavelengths.

Detector settings: step size 5 nm, PMT 12 , Response time 4 s



Figure 34 Characterization of a pure compound from a fluorescence scan



Procedure II - Take two LC runs with the FLD

The conditions for the separation of organic compounds such as polyaromatic nuclear hydrocarbons (PNAs) are well described in various standard methods, including commonly used EPA and DIN methods. Achieving the best detection levels requires checking for the optimum excitation and emission wavelengths for all compounds. Yet taking fluorescence scans individually makes this a tedious process. A better approach is to acquire spectra online for all compounds during a run. This speeds up method development tremendously. Two runs are sufficient for optimization.

During the first run, one wavelength is chosen in the low UV range for the excitation wavelength and one emission wavelength in the spectral range for the emission wavelength. Most fluorophores show strong absorption at these wavelengths and the quantum yield is high. Excitation is sufficient for collecting emission spectra.

Table on page 90 contains all emission spectra obtained in a single run from a mix of 15 PNAs. This set of spectra is used to set up a timetable for optimum emission wavelengths for all compounds.

The individual compound spectra in the isofluorescence plot show that at least three emission wavelengths are needed to detect all 15 PNAs properly:

In the second run, three setpoints for emission wavelengths are entered into the time- program and excitation spectra are recorded, as shown in Figure 36 on page 91. The area of high intensity (red) is caused by stray light when emission spectra overlap with the excitation wavelength. This can be avoided by fitting the spectral range automatically. Excitation at 260 nm is most appropriate for all PNAs.

Table 10 Timetable for PNA analysis

0 min: 350 nm for naphthalene to phenanthrene

8.2 min: 420 nm for anthracene to benzo(g,h,i)perylene

19.0 min: 500 nm for indeno(1,2,3-c,d)pyrene



Table 11 Conditions for Optimization of PNA analysis according to figures below

Column Vydac, 2.1 x 200 mm, PNA, 5 µm

Mobile phase A = water; B = acetonitrile (50 : 50)

Gradient 3 minutes, 60% 14 minutes, 90% 22 minutes, 100%


Column temperature 18 °C


FLD settings PMT 12, response time 4 s, step size 5 nm



This shows the isofluorescence plot of emission spectra for 15 PNAs (5 µg/ml) with a fixed excitation wavelength (260 nm).

Figure 35 Optimization of the time-program for the emission wavelength



Figure 36 Optimization of the time-program for the excitation wavelength

The obtained data are combined to setup the timetable for the excitation wavelength for best limit of detection and selectivity. The optimized switching events for this example are summarized in Table 12 on page 91.

This timetable gives the conditions for optimum detection based on the results of two chromatographic runs.

Table 12 Timetable for the analysis of 15 polynuclear aromatic hydrocarbons

Time [min] Exitation Wavelength [nm] Emission Wavelength [nm]

0 260 350

8.2 260 420

19.0 260 500



Procedure III - Make a single run with a DAD/FLD combination

For most organic compounds, UV- spectra from diode array detectors are nearly identical to fluorescence excitation spectra. Spectral differences are caused by specific detector characteristics such as spectral resolution or light sources.

In practice, combining a diode array detector with a fluorescence detector in series gives the full data set needed to achieve the optimum fluorescence excitation and emission wavelengths for a series of compounds in a single run. With the UV/Visible/excitation spectra available from the diode array detector, the fluorescence detector is set to acquire emission spectra with a fixed excitation wavelength in the low UV range.

The example is taken from the quality control of carbamates. Samples are analyzed for the impurities 2,3- diaminophenazine (DAP) and 2- amino- 3- hydroxyphenazine (AHP). Reference samples of DAP and AHP were analyzed with diode array and fluorescence detection. Table on page 93 shows the spectra obtained from both detectors for DAP. The excitation spectrum of DAP is very similar to the UV absorption spectrum from the diode array detector. Table on page 94 shows the successful application of the method to a carbamate sample and a pure mixture of DAP and AHP for reference. The column was overloaded with the non- fluorescent carbamate (2- benzimidazole carbamic acid methylester/MBC) to see the known impurities, AHP and DAP.



This is an impurity of carbamates. The excitation spectrum in a second run shows the equivalence of UV-spectra and fluorescence excitation spectra. An excitation wavelength at 265 nm was used for taking the emission spectrum and an emission wavelength at 540 nm was used for taking the excitation spectrum.

Figure 37 UV-spectrum and fluorescence spectra for 2,3-diaminophenazine (DAP)



The two upper traces are obtained using two different excitation wavelengths.The lower trace is a pure standard of the known impurities.

Figure 38 Qualitive analysis of MBC (2-benzimidazole carbamic acid methylester) and impurities

Table 13 Conditions for analysis of DAP and MBC according to figures above

Column Zorbax SB, 2 x 50 mm, PNA, 5 µm

Mobile phase A = water; B = acetonitrile

Gradient 0 minutes, 5% 10 minutes, 15%




FLD settings PMT 12, response time 4 s, step size 5 nmEx 265 nm and 430 nmEm 540 nm



Step 3: Set up Routine Methods

In routine analysis, sample matrices can have a significant influence on retention times. For reliable results, sample preparation must be thorough to avoid interferences or LC methods must be rugged enough. With difficult matrices, simultaneous multi- wavelength detection offers more reliability than timetable- controlled wavelength switching. The FLD can, in addition, acquire fluorescence spectra while it records the detector signals for quantitative analysis. Therefore qualitative data are available for peak confirmation and purity checks in routine analysis.

Multi wavelength detection

Time- programmed wavelength switching traditionally is used to achieve low limits of detection and high selectivity in routine quantitative analysis. Such switching is difficult if compounds elute closely and require a change in excitation or emission wavelength. Peaks can be distorted and quantitation made impossible if wavelength switching occurs during the elution of a compound. Very often this happens with complex matrices, influencing the retention of compounds.

In spectral mode, the FLD can acquire up to four different signals simultaneously. All of them can be used for quantitative analysis. Apart from complex matrices, this is advantageous when watching for impurities at additional wavelengths. It is also advantageous for reaching low limits of detection or increasing selectivity through optimum wavelength settings at any time. The number of data points acquired per signal is reduced and thus limits of detection may be higher, depending on the detector settings compared to the signal mode.

PNA analysis, for example, can be performed with simultaneous multi wavelength detection instead of wavelength- switching. With four different wavelengths for emission, all 15 PNAs can be monitored (Table on page 97).



Table 14 Conditions for simultanoeus multi wavelength detection for PNA-analysis (see figure below)

Column Vydac, 2.1 x 250 mm, PNA, 5 µm

Mobile phase A = water; B = acetonitrile (50 : 50 )

Gradient 3 min, 60 %14.5 min, 90 %22.5 min, 95 %

Flow rate 0.4 mL/min


Injection volume 2 µL

FLD settings PMT 12 , response time 4 s



Previously, only diode array detectors and mass spectrometric detectors could deliver spectral information online to confirm peak identity as assigned by retention time.

Now, fluorescence detectors provide an additional tool for automated peak confirmation and purity control. No additional run is necessary after the quantitative analysis.

During method development, fluorescence excitation and emission spectra are collected from reference standards and entered into a library- at the choice of the method developer. All spectral data from unknown samples can then be compared automatically with library data. Table 15 on page 98 illustrates this principle using a PNA analysis. The match factor given in the report for each peak indicates the degree of similarity between the reference spectrum and the spectra from a peak. A match factor of 1,000 means identical spectra.

The upper trace was received with traditional wavelength switching.

Figure 39 Simultaneous multi wavelength detection for PNA-analysis



In addition, the purity of a peak can be investigated by comparing spectra obtained within a single peak. When a peak is calculated to be within the user- defined purity limits, the purity factor is the mean purity value of all spectra that are within the purity limits.

The reliability of the purity and the match factor depends on the quality of spectra recorded. Because of the lower number of data points available with the fluorescence detector in general, the match factors and purity data obtained show stronger deviations compared to data from the diode array detector, even if the compounds are identical.

Table 15 on page 98 shows an automated library search based on the emission spectra from a PNA reference sample.

Table 15 Peak confirmation using a fluorescence spectral library

Meas. RetTime

Library CalTbl Signal Amount Purity # Match Libary Name

[min] [min] [min] [ng] Factor

4.859 4.800 5.178 1 1.47986e-1 - 1 993 Naphthalene@em

6.764 7.000 7.162 1 2.16156e-1 - 1 998 Acenaphthene@em

7.137 7.100 7.544 1 1.14864e-1 - 1 995 Fluorene@em

8.005 8.000 8.453 1 2.56635e-1 - 1 969 Phenanthrene@em

8.841 8.800 9.328 1 1.76064e-1 - 1 993 Anthracene@em

9.838 10.000 10.353 1 2.15360e-1 - 1 997 Fluoranthene@em

10.439 10.400 10.988 1 8.00754e-2 - 1 1000 Pyrene@em

12.826 12.800 13.469 1 1.40764e-1 - 1 998 Benz(a)anthracene@em

13.340 13.300 14.022 1 1.14082e-1 - 1 999 Chrysene@em

15.274 15.200 16.052 1 6.90434e-1 - 1 999 Benzo(b)fluoranthene@em

16.187 16.200 17.052 1 5.61791e-1 - 1 998 Benzo(k)fluoranthene@em

16.865 16.900 17.804 1 5.58070e-1 - 1 999 Benz(a)pyrene@em

18.586 18.600 19.645 1 5.17430e-1 - 1 999 Dibenz(a,h)anthracene@em

19.200 19.100 20.329 1 6.03334e-1 - 1 995 Benzo(g,h,i)perylene@em

20.106 20.000 21.291 1 9.13648e-2 - 1 991 Indeno(1,2,3-c,d)pyrene@em


Using the Fluorescence Detector 4Example: Optimization for Multiple Compounds

Example: Optimization for Multiple Compounds

Example: Optimization for Multiple Compounds

Using PNAs as a sample, this example uses the described scanning functions.


4 Using the Fluorescence DetectorExample: Optimization for Multiple Compounds

Setting the Chromatographic Conditions

This example uses the following chromatographic conditions (the detector settings are shown in Figure 40 on page 101).

Table 16 Chromatographic Conditions

Mobile phases A = water = 50 % B = Acetonitrile = 50 %

Column Vydac-C18-PNA, 250 mm x 2.1 mm i.d. with 5 µm particles

Sample PAH 0.5 ng


Compressibility A (water) 46

Compressibility B (Acetonitrile) 115

Stroke A and B auto

Time Table at 0 min % B=50

at 3 min % B=60

at 14.5 min % B=90

at 22.5 min % B=95

Stop time 26 min

Post time 8 min


Oven temperature (1200) 30 °C

FLD PMT Gain PMT = 15

FLD Response time 4 s



1 Wait until the baseline stabilizes. Complete the run.

Select a Excitation wavelength in the low UV (230...260 nm). This will cover nearly all fluorescence in your sample.

DO NOT select additional emission wavelengths (B, C, D).Doing so will increase the scan time and will lower the performance.

Figure 40 Detector Settings for Emission Scan



2 Load the signal. (In this example just the time range of 13 min is displayed).

Figure 41 Chromatogram from Emissions Scan



3 Use the isoabsorbance plot and evaluate the optimal emission wavelengths, shown in the table below.

Figure 42 Isoabsorbance Plot from Emission Scan

Table 17

Peak # Time Emission Wavelength

1 5.3 min 330 nm

2 7.2 min 330 nm

3 7.6 min 310 nm

4 8.6 min 360 nm

5 10.6 min 445 nm

6 11.23 min 385 nm



4 Using the settings and the timetable (from previous page), do a second run for the evaluation of the optimal excitation wavelength. See Figure 43 on page 104.

5 Wait until the baseline stabilizes. Start the run.

DO NOT select additional excitation wavelengths (B, C, D).Doing so will increase the scan time and will lower the performance.

Figure 43 Detector Settings for Excitation Scan



6 Load the signal.

Figure 44 Chromatogram - Excitation Scan at Reference Wavelength 260/330 nm

7 Use the isoabsorbance plot and evaluate the optimal excitation wavelengths (in this example just in the time range of 13 minutes).

Figure 45 Isoabsorbance Plot - Excitation

The table below shows the complete information about emission (from Figure 42 on page 103) and excitation maxima.



Table 18

Peak # Time Emission Wavelength Excitation Wavelength

1 5.3 min 330 nm 220 / 280 nm

2 7.3 min 330 nm 225 / 285 nm

3 7.7 min 310 nm 265 nm

4 8.5 min 360 nm 245 nm

5 10.7 min 445 nm 280 nm

6 11.3 min 385 nm 270 / 330 nm



Evaluating The System Background

The example below uses water.

1 Pump solvent through your system.

2 Set the fluorescence scan range under FLD special setpoints according to your needs.

3 Set PMT gain to 16.

NOTE The scan time will increase when the range is enlarged. With the default values, the scan takes about 2 minutes.

The wavelength range and step number defines the duration. Using the maximum range, the scan would take approximately 10 minutes.

Figure 46 FLD Special Settings



4 Define a data file name and take a fluorescence scan. After the scan is completed, the isoabsorbance scan results appear, see Figure 47 on page 108.

Figure 47 Fluorescence Scan of Water

NOTE A low background will improve the signal-to-noise, see also “Reducing Stray Light” on page 135.


Using the Fluorescence Detector 4How to collect spectra with modes SPECTRA ALL IN PEAK and APEX SPECTRA ONLY

How to collect spectra with modes SPECTRA ALL IN PEAK and APEX SPECTRA ONLY

This section describes how to overcome a malfunction in the current implementation of the Agilent ChemStation with the Fluorescence Detector (G1321A/B). In these modes spectra intermittently are not collected into the data file.

The peak triggered spectra acquisition in the FLD is controlled by 2 parameters - THRS (Threshold) and PDPW (PeakDetector PeakWidth). In addition the parameter PKWD (Detector PeakWidth) only influences the filtering of the chromatogram.

1 Set the parameters THRS, PDPW and PKWD accordingly to the current chromatogram.

Best results for collecting peak triggered spectra are gathered when PDPW is 2 steps lower than PKWD, see “Peakwidth Settings” on page 134.


4 Using the Fluorescence DetectorHow to collect spectra with modes SPECTRA ALL IN PEAK and APEX SPECTRA ONLY

2 In the FLD’s setup- screen there are 2 fields to enter the PKWD Peakwidth (Responsetime) and the THRS Threshold (visible when Multi-EX or Multi-EM is selected). Defaults are: PKWD = 6 (0.2 min); THRS = 5.000 LU.


Using the Fluorescence Detector 4How to collect spectra with modes SPECTRA ALL IN PEAK and APEX SPECTRA ONLY

The selected values are fixed during the run. Changes of PDPW are only possible using the Peakwidth- field in the Timetable (visible when Multi-EX or Multi-EM is selected).

THRS and PDPW influence the peak- triggered spectra- acquisition. You can change THRS in the FLD’s setup- screen; PDPW can only be changed with the Peakwidth- field in the Timetable.

NOTE When you change PKWD you should change PDPW also. Enter in the Timetable at 0.0 min a PDPW = PKWD - 2 (e.g. PKWD = 0.2 min, PDPW = 0.05 min). In case of a longer chromatogram and peak broadening later on you can increase the PDPW-value by 1 step with an additional entry in the Timetable.


4 Using the Fluorescence DetectorHow to collect spectra with modes SPECTRA ALL IN PEAK and APEX SPECTRA ONLY

Notes:

• The peak- detection algorithm works best when a peak is reduced to 8 – 16 data points. The FLD collects the data points with an internal data rate of 74.08 Hz (= 13.50 ms) (1 signal only). The data reduction is only influenced by the PDPWparameter. When PDPW is too low, the peakdetector does not find a peak, instead it assumes a rising/falling baseline at begin/end of the peak. In case PDPW is too big the peakdetector assumes that the peak is noise.

• The peakdetector works online on the current chromatogram. This means that begin/apex/end of a peak is recognized with delay. Additionally the points of spectra are sequentially acquired. This means that the acquisition of wide range spectra lasts much longer than the acquisition of a short- range spectrum. When you have a fast chromatography it is nearly impossible to collect a "clean" APEX- spectra: the first/last points of the spectra are acquired before/after you have the highest concentration in the detector’s cell.

• How long the acquisition of single spectra lasts is shown in the FLD’s setup screen.


Using the Fluorescence Detector 4Solvent Information

Solvent Information

Observe the following recommendations on the use of solvents.

• Follow recommendations for avoiding the growth of algae, see pump manuals.

• Small particles can permanently block capillaries and valves. Therefore, always filter solvents through 0.4 µm filters.

• Avoid or minimize the use of solvents that may corrode parts in the flow path. Consider specifications for the pH range given for different materials like flow cells, valve materials etc. and recommendations in subsequent sections.

Material Information

Materials in the flow path are carefully selected based on Agilent’s experiences in developing highest quality instruments for HPLC analysis over several decades. These materials exhibit excellent robustness under typical HPLC conditions. For any special conditions, please consult the material information section or contact Agilent.

Disclaimer

Subsequent data were collected from external resources and are meant as a reference. Agilent cannot guarantee the correctness and completeness of such information. Data is based on compatibility libraries, which are not specific for estimating the long- term life time under specific but highly variable conditions of UHPLC systems, solvents, solvent mixtures and samples. Information can also not be generalized due to catalytic effects of impurities like metal ions, complexing agents, oxygen etc. Apart from pure chemical corrosion, other effects like electro corrosion, electrostatic charging (especially for non- conductive organic solvents), swelling of polymer parts etc. need to be considered. Most data available refers to room temperature (typically 20 – 25 °C, 68 – 77 °F). If corrosion is possible, it usually accelerates at higher temperatures. If in doubt, please consult technical literature on chemical compatibility of materials.


4 Using the Fluorescence DetectorSolvent Information

PEEK

PEEK (Polyether- Ether Ketones) combines excellent properties regarding biocompatibility, chemical resistance, mechanical and thermal stability. PEEK is therefore the material of choice for UHPLC and biochemical instrumentation.

It is stable in a wide pH range, and inert to many common solvents.

There is still a number of known incompatibilities with chemicals such as chloroform, methylene chloride, THF, DMSO, strong acids (nitric acid > 10 %, sulphuric acid > 10 %, sulfonic acids, trichloroacetic acid), halogenes or aequous halogene solutions, phenol and derivatives (cresols, salicylic acid etc.).

Polyimide

Agilent uses semi- crystalline polyimide for rotor seals in valves and needle seats in autosamplers. One supplier of polyimide is DuPont, which brands polyimide as Vespel, which is also used by Agilent.

Polyimide is stable in a pH range between 1 and 10 and in most organic solvents. It is incompatible with concentrated mineral acids (e.g. sulphuric acid), glacial acetic acid, DMSO and THF. It is also degraded by nucleophilic substances like ammonia (e.g. ammonium salts in basic conditions) or acetates.

Polyethylene (PE)

Agilent uses UHMW (ultra- high molecular weight)- PE/PTFE blends for yellow piston and wash seals, which are used in 1290 Infinity pumps and for normal phase applications in 1260 Infinity pumps.

Polyethylene has a good stability for most common inorganic solvents including acids and bases in a pH range of 1 to 12.5 . It is compatible to many organic solvents used in chromatographic systems like methanol, acetonitrile and isopropanol. It has limited stability with aliphatic, aromatic and halogenated hydrocarbons, THF, phenol and derivatives, concentrated acids and bases. For normal phase applications, the maximum pressure should be limited to 200 bar.



Tantalum (Ta)

Tantalum is inert to most common HPLC solvents and almost all acids except fluoric acid and acids with free sulfur trioxide. It can be corroded by strong bases (e.g. hydroxide solutions > 10 %, diethylamine). It is not recommended for the use with fluoric acid and fluorides.

Stainless Steel (ST)

Stainless steel is inert against many common solvents. It is stable in the presence of acids and bases in a pH range of 1 to 12.5 . It can be corroded by acids below pH 2.3 . It can also corrode in following solvents:

• Solutions of alkali halides, their respective acids (for example, lithium iodide, potassium chloride, and so on) and aqueous solutions of halogens.

• High concentrations of inorganic acids like nitric acid, sulfuric acid and organic solvents especially at higher temperatures (replace, if your chromatography method allows, by phosphoric acid or phosphate buffer which are less corrosive against stainless steel).

• Halogenated solvents or mixtures which form radicals and/or acids, for example:

2 CHCl3 + O2→ 2 COCl2 + 2 HCl

This reaction, in which stainless steel probably acts as a catalyst, occurs quickly with dried chloroform if the drying process removes the stabilizing alcohol.

• Chromatographic grade ethers, which can contain peroxides (for example, THF, dioxane, di- isopropylether). Such ethers should be filtered through dry aluminium oxide which adsorbs the peroxides.

• Solutions of organic acids (acetic acid, formic acid, and so on) in organic solvents. For example, a 1 % solution of acetic acid in methanol will attack steel.

• Solutions containing strong complexing agents (for example, EDTA, ethylene diamine tetra- acetic acid).

• Mixtures of carbon tetrachloride with 2- propanol or THF.



Diamond-Like Carbon (DLC)

Diamond- Like Carbon is inert to almost all common acids, bases and solvents. There are no documented incompatibilities for HPLC applications.

Fused silica and Quartz (SiO2)

Fused silica is used in 1290 Infinity Flow Cells and capillaries. Quartz is used for classical flow cell windows. It is inert against all common solvents and acids except hydrofluoric acid and acidic solvents containing fluorides. It is corroded by strong bases and should not be used above pH 12 at room temperature. The corrosion of flow cell windows can negatively affect measurement results. For a pH greater than 12, the use of flow cells with sapphire windows is recommended.

Gold

Gold is inert to all common HPLC solvents, acids and bases within the specified pH range. It can be corroded by complexing cyanides and concentrated acids like aqua regia.

Zirconium Oxide (ZrO2)

Zirconium Oxide is inert to almost all common acids, bases and solvents. There are no documented incompatibilities for HPLC applications.

Platinum/Iridium

Platinum/Iridium is inert to almost all common acids, bases and solvents. There are no documented incompatibilities for HPLC applications.

Fluorinated polymers (PTFE, PFA, FEP, FFKM)

Fluorinated polymers like PTFE (polytetrafluorethylene), PFA (perfluoroalkoxy) and FEP (fluorinated ethylene propylene) are inert to almost all common acids, bases, and solvents. FFKM is perfluorinated rubber, which is also resistant to most chemicals. As an elastomer, it may swell in some organic solvents like halogenated hydrocarbons.

TFE/PDD copolymer tubings, which are used in all Agilent degassers except G1322A, are not compatible with fluorinated solvents like Freon, Fluorinert, or Vertrel. They have limited life time in the presence of



Hexafluoroisopropanol (HFIP). To ensure the longest possible life with HFIP, it is best to dedicate a particular chamber to this solvent, not to switch solvents, and not to let dry out the chamber. For optimizing the life of the pressure sensor, do not leave HFIP in the chamber when the unit is off.

Sapphire, Ruby and Al2O3-based ceramics

Sapphire, ruby and ceramics based on aluminum oxide Al2O3 are inert to almost all common acids, bases and solvents. There are no documented incompatibilities for HPLC applications.



5Optimizing the Detector

Optimization Overview 120

Design Features Help Optimization 122

Check Performance Before You Start 122

Finding the Best Wavelengths 123

A Real Example 124

Finding the Best Signal Amplification 125

FLD Scaling Range and Operating Conditions 126

Changing the Xenon Flash Lamp Frequency 131

Lamp Life Savings 132

Selecting the Best Response Time 133

Reducing Stray Light 135

This chapter provides information on how to optimize the detector.


5 Optimizing the DetectorOptimization Overview

Optimization Overview

1 Setting the right PMT value

For most applications a setting of 10 is adequate (see “Finding the Best Signal Amplification” on page 125). The FLD A/D converter exhibits a large linear range making PMT switching unnecessary for most applications. For example, if at high concentrations a peak is cut off; decrease the PMT setting. Remember that low PMT settings decrease the signal to noise ratio.

The built- in PMT gain test uses the parameters in the detector. When using the PMT gain test, the wavelength setting and lamp energy mode (depending on Multiwavelength- Mode and Lamp- Economy) will affect the PMT gain calculation.

2 Using an appropriate response time

For most applications a setting of 4 seconds is adequate (see “Selecting the Best Response Time” on page 133). Only for high speed analyses (short columns at high flow rates) a lower setting is recommended. Bear in mind that even if the response time is too high fast peaks will appear a little smaller and broader but retention time and peak areas are still correct and reproducible.

NOTE Some features (e.g. spectrum acquisition, multi-wavelength detection) described in this chapter are not available on the G1321C 1260 Infinity Fluorescence Detector.

NOTE PMT Gain Test

The PMT Gain test was provided in the classic Agilent ChemStation within the settings of the FLD. It is not available in the Agilent OpenLAB CDS and G4208A Instant Pilot.

The PMT Gain test has been incorporated into in the Agilent Lab Advisor B.02.04 [093]. The PMT Gain Test can be found under Instrument Control > Special Commands.

NOTE If you have changed one or more parameter(s), you have to press 'OK' to write down the new settings into the FLD. Then re-enter 'FLD-Signals' and start the PMT gain test.


Optimizing the Detector 5Optimization Overview

3 Finding the optimum wavelength

Most fluorescent active molecules absorb at 230 nm (see “Finding the Best Wavelengths” on page 123). Set the excitation wavelength to 230 nm and online scan the emission spectra (multi- emission mode). Then set the determined emission wavelength and perform a multi- excitation scan (multi- excitation mode) to find the best excitation wavelength.

4 Evaluating fluorescence spectra

In contrast to diode array based UV detectors where UV spectra are evaluated by taking a spectrum at the peak maximum and selecting a reference spectrum at the baseline, correct fluorescence spectra are obtained by selecting a peak maximum spectrum and a reference around the inflection points. Selecting reference spectra at the baseline is not useful because the spectrum on the baseline is very noisy (no light!).

5 Switching lamp ON only for analysis

Unless maximum sensitivity is needed, the lamp lifetime can significantly be increased by switching it on just for analysis. In contrast to other LC detectors the fluorescence detector equilibrates within seconds after the lamp is switched ON.

6 Do not overpressurize the detector flow cell

Be aware to not exceed a 20 bar pressure drop after the flow cell when hooking up additional devices like other detectors or a fraction collector. It's better to place a UV detector before the fluorescence detector.

NOTE For highest reproducibility and linearity change the lamp setting to always ON (default is on only during run).

One hour of initial warm-up of the instrument is recommended.

NOTE When comparing fluorescence excitation spectra directly with DAD spectra or literature based absorbance spectra, you should consider large differences in the used optical bandwidth (FLD = 20 nm) which cause a systematic wavelength maximum shift depending on the absorbance spectrum of the compound under evaluation.


5 Optimizing the DetectorDesign Features Help Optimization

Design Features Help Optimization

The module has several features you can use to optimize detection:

Check Performance Before You Start

Before you start you should check that your detector is performing according to the specifications published by Agilent Technologies.

Your normal LC grade solvents may give good results most of the time but our experience shows that baseline noise can be higher with LC grade solvents than with fluorescence grade solvents.

Flush your solvent delivery system for at least 15 minutes before checking sensitivity. If your pump has multiple channels, you should also flush the channels not in use.

PMTGAIN Amplification factor

LAMP Flash frequency

RESPONSETIME Data reduction interval


Optimizing the Detector 5Finding the Best Wavelengths

Finding the Best Wavelengths

The most important parameters to be optimized in fluorescence detection are the excitation and emission wavelengths. Generally, it is assumed that the best excitation wavelength can be taken from the excitation spectrum acquired on a spectrofluorimeter. It is also assumed that once the optimal excitation wavelength has been found for one particular instrument type this wavelength can also be applied to other instruments.

Both assumptions are wrong.

The optimum wavelength for the excitation depends on the absorption of the compounds but also on the instrument characteristics, for example the lamp type and the gratings. As most organic molecules absorb best in the ultraviolet range the module was designed to give an optimum signal- to- noise ratio in the 210 nm to 360 nm range of the spectrum. To achieve greatest sensitivity, the absorbance wavelength of your sample molecule should match the wavelength range for your instrument. In other words, an excitation wavelength in the ultraviolet range. Your module has a broad excitation wavelength range, but for higher sensitivity you should choose a wavelength in the ultraviolet range (near 250 nm).

The design elements that contribute to lower efficiency in the lower ultraviolet range are the xenon flash lamp and the gratings. Flash- type lamps shift the optimum wavelength to lower wavelength ranges with the module to a maximum of 250 nm. The excitation grating is blazed for highest efficiency at 300 nm.


5 Optimizing the DetectorFinding the Best Wavelengths

A Real Example

Although an excitation wavelength of 340 nm is quoted in the literature the module scan of orthophthalaldehyde, a derivative of the amino acid alanine, ( Figure 48 on page 124) shows a maximum between 220 nm and 240 nm.

Figure 48 Scan Orthophthalaldehyde Derivative of Alanine

When you are looking for the wavelength by scanning, scan over the whole range. As this example shows a maximum may be found in a completely different wavelength range.

NOTE When comparing fluorescence excitation spectra directly with DAD spectra or literature based absorbance spectra, you should consider large differences in the used optical bandwidth (FLD = 20 nm) which cause a systematic wavelength maximum shift depending on the absorbance spectrum of the compound under evaluation.


Optimizing the Detector 5Finding the Best Signal Amplification

Finding the Best Signal Amplification

Increasing the PMTGAIN increases the signal and the noise. Up to a certain factor the increase in signal is higher than the increase in noise.

The step from gain to gain is equal to a factor of 2 (which is the same as on the HP 1046A FLD).

In Figure 49 on page 125 the PMTGAIN was gradually raised from 4 up to 11 (the peak is from the Agilent Technologies isocratic sample which was diluted 1000 times). With increasing PMTGAIN there was an improvement in signal- to- noise up to 10. Above 10 the noise increased proportionately to the signal with no improvement in signal- to- noise.

Figure 49 Finding Best PMTGAIN for Biphenyl

The reason for this is the fact, that quantification of baselines (especially at low background levels) is not sufficient for statistically working filter methods. For the best gain, check your solvent under flow conditions with the auto- gain function. Do not use higher values than proposed by the system, if not necessary, because of excessive high fluorescence signals.

Use the PMT test to automatically determine the setting.


5 Optimizing the DetectorFinding the Best Signal Amplification

FLD Scaling Range and Operating Conditions

When using different FLD

• The signal height of individual G1321 FLD modules may exceed the recommended signal range 0 – 100 LU. Under certain circumstances this could lead to clipped peaks.

• Different G1321 FLD modules show different signal heights with identical methods. This is not a problem in general but could be inconvenient when operating more than one G1321 FLD in the lab.

Both scaling issues can be resolved. Refer to “Optimize the PMT- Gain- Level” on page 126.

Optimize the PMT-Gain-Level

Start the PMT- Gain- Test with your operating conditions (used method parameter, EX- /EM- wavelength, solvent, flow rate, …). The resulting PMT value will give you the best signal to noise performance with the maximum usable signal range for this method and this specific instrument. For another FLD this PMT level may vary (based on the individual PMT- Gain- Test).

The figure below demonstrates the impact of changing the PMT Gain.



Figure 50 PMT Gain Behavior

In this example the maximum output is around 220 LU and further increase of the PMT (above 9) results in a signal overload (clipping) and drop of signal to noise value.

1 Set the PMT- Gain Level

Now check with your highest concentration amount, that your highest peak does not clip or overflow.

• If this check is ok, you finished the PMT- Gain- Level optimization. Continue with "Set your Luminescence Units in LU".

• If the check shows that the highest concentration doesn't fit to the selected range (e.g. by clipping), you may decrease the sensitivity of your FLD by gradually decreasing the PMT- Level by 1 to get roughly half the signal height by each step. Be aware that by that step you will lose sensitivity at low signal levels (LOD).



2 Set your Luminescence Units in LU

If you are not satisfied with the LU output level of the detector or if you want to align the output of multiple instruments with different output levels you can scale each instrument output.

The recommended setting of the G1321 FLD is around 100 LU for the highest peak height to get optimum signal to noise and signal range. Lower LU values normally do not influence the performance of the instrument if PMT- Gain Test was executed fine.

For analog out less than 100 LU is optimum to get best analog signal performance with the default attenuation of 100 LU/ 1 V. Adapt your LU setting such that your maximum signal level under default attenuation is between 50 to 80 LU (analog output equivalent to 500 mV to 800 mV).

After correct PMT Setting you can scale any instrument to your favorable LU level. We recommend not exceeding around 100 LU. The parameter of choice is called 'Scale factor' and is applicable by the local controller, the Instant Pilot (B.02.07 or later).

In case older revisions are used, the 'Scale factor' can be entered using the command line of

• Agilent ChemStation: PRINT SENDMODULE$(LFLD,"DMUL x.xx")

• Instant Pilot: Service Mode – FLD, then type DMUL x.xx and press SEND.

• LAN/RS- 232 Firmware Update Tool: via Send Instruction menu: DMUL x.xx

• Agilent Lab Advisor Software: via Instruction menu: DMUL x.xx

This setting is resident to the instrument even for firmware updates and is independent of the software environment.

The level of LU is no measure of instrument sensitivity! At the lowest concentration limit (limit of detection), the signal to noise (e.g. by Raman S/N Test) is the only measure that can accurately be used to compare chromatograms and results and to confirm the performance of the instrument.

For low background and highest sensitivity keep the flow cell clean and use always fresh water to prevent biological background from native fluorescence by algae and bacteria.



Visualization of ADC Limits

A new firmware (A.06.11) for the Fluorescence Detector G1321A/B has been released that includes a new feature, the "Visualization of ADC Limits".

Up to firmware A.06.10, an "ADC overflow" was not visible in the chromatogram under certain method conditions.

Overflow could be concealed by smoothing of a filter and thus not visible for the user. In the Agilent ChemStation, the "ADC overflow" event was only shown in the logbook.

This problem did only occur if the Peakwidth (Responsetime) parameter has been set similar or larger than the real width of the chromatographic peak.



As a result, the "ADC overflow" is visible as a real flat peak in the chromatogram showing the user, that the setting of the detector parameter (PMT gain or the concentration of the solution) is set to high.

Raw ADC countsThe measured light intensity is limited by the max range of the ADC-converter.

A filter smoothes the peak making it not clearly visible that the max intensity is reached. Also peak area and peak height are distorted which leads to poor linearity performance.Note that "max LU" is not a fix number but depends on the intensity of the reference channel!

New implementation (with firmware A.06.11 or above)While any sample value within the filter width is in state "ADC overflow" the max possible LU is displayed in chromatogram.Note that "max LU" is slightly dependent on lamp drift and lamp noise but strongly dependent on the excitation wavelength.

NOTE The transfer of methods 1:1 from one FLD to another may result into the above "ADC overflow" problem. For details see “FLD Scaling Range and Operating Conditions” on page 126“FLD Scaling Range and Operating Conditions”.


Optimizing the Detector 5Changing the Xenon Flash Lamp Frequency

Changing the Xenon Flash Lamp Frequency

Modes

The lamp flash frequency can be changed into the following modes:

Best sensitivity can be expected with “no economy”, see Figure 51 on page 131.

Figure 51 Xenon Flash Lamp Frequency

Table 19 Flash Lamp Modes

Positioning 296 Hz (Standard), 560 V 63 mJ (18.8 W)

74 Hz (Economy), 560 V 63 mJ (4.7 W)

Rotation (Multi Ex/Em) 74 Hz (Standard), 950 V 180 mJ (13.3 W)

74 Hz (Economy), 560 V 63 mJ (4.7 W)


5 Optimizing the DetectorChanging the Xenon Flash Lamp Frequency

Lamp Life Savings

There are three ways to save lamp life:

• switch to “lamp on during run” without loss of sensitivity.

• switch to “economy” mode with a certain loss of sensitivity.

• a combination of the above.


Optimizing the Detector 5Selecting the Best Response Time

Selecting the Best Response Time

Selecting the Best Response Time

Data reduction using the RESPONSETIME function will increase your signal- to- noise ratio.

For example, see Figure 52 on page 133.

Figure 52 Finding Best Response Time

LC fluorescence detectors typically work with response times of 2 or 4 s. The default of the module is 4 seconds. It is important to know that comparing sensitivity requires using the same response time. A response time of 4 s (default) is equivalent to a time constant of 1.8 s and appropriate for standard chromatographic conditions.

Figure 53 Separation of Peaks using Responsetime


5 Optimizing the DetectorSelecting the Best Response Time

Peakwidth Settings

Peakwidth enables you to select the peak width (response time) for your analysis. The peak width is defined as the width of a peak, in minutes, at half the peak height. Set the peak width to the narrowest expected peak in your chromatogram. The peak width sets the optimum response time for your detector. The peak detector ignores any peaks that are considerably narrower, or wider, than the peak width setting. The response time is the time between 10 % and 90 % of the output signal in response to an input step function.

Limits: When you set the peak width (in minutes), the corresponding response time is set automatically and the appropriate data rate for signal and spectra acquisition is selected as shown in the table below.

NOTE Do not use peak width shorter than necessary.

Table 20 Peakwidth Setting

Peak Width Data Rate

At half height [min]

Response [sec]

Hz ms

> 0.0016 0.016 144.93 6.9 G1321B, K1321B(with firmware A.06.54 and above)

< 0.003 0.03 74.07 13.5 G1321B/C, K1321B

> 0.003 0.06 37.04 27.0

G1321A/B/C, K1321B

> 0.005 0.12 37.04 27.0

> 0.01 0.25 37.04 27.0

> 0.025 0.5 18.52 54.0

> 0.05 1.0 9.26 108.0

> 0.1 2.0 4.63 216.0

> 0.2 4.0 2.31 432.0

> 0.4 8.0 1.16 864.0


Optimizing the Detector 5Reducing Stray Light

Reducing Stray Light

Cut- off filters are used to remove stray light and 2nd order or higher stray light by allowing complete transmission above the cut- off and little or no transmission below the cut- off point. They are used between excitation and emission gratings, to prevent any stray excitation light from reaching the photomultiplier tube, when it is measuring emission.

When the emission and excitation wavelengths are close together, the distortion due to scattering severely limits the sensitivity. When the

emission wavelength is twice the excitation wavelength the 2nd order light is the limiting factor. To explain the effect of such higher order light, assume the detector is on, but no sample is eluting through the flow cell.

The lamp sends 1 million photons into the flow cell at, for example 280 nm. Scattering on the surface of the flow cell and scattering from the molecules of solvent allow 0.1 % of this light to leave the cell through the window at right angles to the incident light. Without a cut- off filter, these remaining 1000 photons will reach the emission grating. 90 % will be reflected totally without dispersion onto the photomultiplier. The other 10

% disperses at 280 nm (1st order) and at 560 nm (2nd order). To remove this stray light, you need a cut- off filter around 280 nm.

Because of a known set of applications a 295 nm cut- off filter is built- in for undisturbed application up to 560 nm without compromises (see Figure 54 on page 136).


5 Optimizing the DetectorReducing Stray Light

Figure 54 Reducing Stray Light



6Troubleshooting and Diagnostics

Overview of the Module’s Indicators and Test Functions 138

Status Indicators 139

Power Supply Indicator 139

Module Status Indicator 140

User Interfaces 141

Agilent Lab Advisor Software 142

This chapter gives an overview about the troubleshooting and diagnostic features and the different user interfaces.


6 Troubleshooting and DiagnosticsOverview of the Module’s Indicators and Test Functions

Overview of the Module’s Indicators and Test Functions

Status Indicators

The module is provided with two status indicators which indicate the operational state (prerun, run, and error states) of the module. The status indicators provide a quick visual check of the operation of the module.

Error Messages

In the event of an electronic, mechanical or hydraulic failure, the module generates an error message in the user interface. For each message, a short description of the failure, a list of probable causes of the problem, and a list of suggested actions to fix the problem are provided (see chapter Error Information).

Test Functions

A series of test functions are available for troubleshooting and operational verification after exchanging internal components (see Tests and Calibrations).

Wavelength Recalibration

Wavelength recalibration is recommended after repair of internal components to ensure correct operation of the detector. The detector uses specific properties of the excitation and emission light characteristics (see “Wavelength Calibration Procedure” on page 177).


Troubleshooting and Diagnostics 6Status Indicators

Status Indicators

Two status indicators are located on the front of the module. The lower left indicates the power supply status, the upper right indicates the module status.

Figure 55 Location of Status Indicators

Power Supply Indicator

The power supply indicator is integrated into the main power switch. When the indicator is illuminated (green) the power is ON.


6 Troubleshooting and DiagnosticsStatus Indicators

Module Status Indicator

The module status indicator indicates one of six possible module conditions:

• When the status indicator is OFF (and power switch light is on), the module is in a prerun condition, and is ready to begin an analysis.

• A green status indicator, indicates the module is performing an analysis (run mode).

• A yellow indicator indicates a not- ready condition. The module is in a not- ready state when it is waiting for a specific condition to be reached or completed (for example, immediately after changing a set point), or while a self- test procedure is running.

• An error condition is indicated when the status indicator is red. An error condition indicates the module has detected an internal problem which affects correct operation of the module. Usually, an error condition requires attention (e.g. leak, defective internal components). An error condition always interrupts the analysis.

If the error occurs during analysis, it is propagated within the LC system, i.e. a red LED may indicate a problem of a different module. Use the status display of your user interface for finding the root cause/module of the error.

• A blinking indicator indicates that the module is in resident mode (e.g. during update of main firmware).

• A fast blinking indicator indicates that the module is in a low- level error mode. In such a case try to reboot the module or try a cold- start (see “Special Settings” on page 242. Then try a firmware update (see “Replacing Module Firmware” on page 198). If this does not help, a main board replacement is required.


Troubleshooting and Diagnostics 6User Interfaces

User Interfaces

Depending on the user interface the available tests vary. All test descriptions are based on the Agilent ChemStation as user interface. Some descriptions are only available in the Service Manual.

Table 21 Test Functions avaible vs. User Interface

Test ChemStation Instant Pilot G4208A Lab Advisor

D/A Converter No No Yes

Test Chromatogram Yes (C) No Yes

Wavelength Calibration

Yes Yes (M) Yes

Lamp Intensity Yes No Yes

Dark Current Yes No Yes

C via command

M section Maintenance

D section Diagnose


6 Troubleshooting and DiagnosticsAgilent Lab Advisor Software

Agilent Lab Advisor Software

The Agilent Lab Advisor software is a standalone product that can be used with or without data system. Agilent Lab Advisor software helps to manage the lab for high quality chromatographic results and can monitor in real time a single Agilent LC or all the Agilent GCs and LCs configured on the lab intranet.

Agilent Lab Advisor software provides diagnostic capabilities for all Agilent 1200 Infinity Series modules. This includes diagnostic capabilities, calibration procedures and maintenance routines for all the maintenance routines.

The Agilent Lab Advisor software also allows users to monitor the status of their LC instruments. The Early Maintenance Feedback (EMF) feature helps to carry out preventive maintenance. In addition, users can generate a status report for each individual LC instrument. The tests and diagnostic features as provided by the Agilent Lab Advisor software may differ from the descriptions in this manual. For details refer to the Agilent Lab Advisor software help files.

The Instrument Utilities is a basic version of the Lab Advisor with limited functionality required for installation, use and maintenance. No advanced repair, troubleshooting and monitoring functionality is included.



7Error Information

What Are Error Messages 145

General Error Messages 146

Timeout 146

Shutdown 146

Remote Timeout 147

Lost CAN Partner 147

Leak 148

Leak Sensor Open 148

Leak Sensor Short 149

Compensation Sensor Open 149

Compensation Sensor Short 150

Fan Failed 150

Detector Error Messages 151

Lamp Cover Open 151

FLF Board not found 151

ADC Not Calibrated 152

A/D Overflow 152

Flash Lamp Current Overflow 153

No light at reference diode despite lamp is on 153

Flash Trigger Lost 154

Wavelength Calibration Failed 154

Wavelength Calibration Lost 155

Flow Cell Removed 155

Motor Errors 156


7 Error InformationAgilent Lab Advisor Software

This chapter describes the meaning of error messages, and provides information on probable causes and suggested actions how to recover from error conditions.


Error Information 7What Are Error Messages

What Are Error Messages

Error messages are displayed in the user interface when an electronic, mechanical, or hydraulic (flow path) failure occurs which requires attention before the analysis can be continued (for example, repair, or exchange of consumables is necessary). In the event of such a failure, the red status indicator at the front of the module is switched on, and an entry is written into the module logbook.

If an error occurs outside a method run, other modules will not be informed about this error. If it occurs within a method run, all connected modules will get a notification, all LEDs get red and the run will be stopped. Depending on the module type, this stop is implemented differently. For example, for a pump the flow will be stopped for safety reasons. For a detector, the lamp will stay on in order to avoid equilibration time. Depending on the error type, the next run can only be started, if the error has been resolved, for example liquid from a leak has been dried. Errors for presumably single time events can be recovered by switching on the system in the user interface.

Special handling is done in case of a leak. As a leak is a potential safety issue and may have occurred at a different module from where it has been observed, a leak always causes a shutdown of all modules, even outside a method run.

In all cases, error propagation is done via the CAN bus or via an APG remote cable (see documentation for the APG interface).


7 Error InformationGeneral Error Messages

General Error Messages

General error messages are generic to all Agilent series HPLC modules and may show up on other modules as well.

Timeout Error ID: 0062

The timeout threshold was exceeded.

ShutdownError ID: 0063

An external instrument has generated a shutdown signal on the remote line.

The module continually monitors the remote input connectors for status signals. A LOW signal input on pin 4 of the remote connector generates the error message.

Probable cause Suggested actions

1 The analysis was completed successfully, and the timeout function switched off the module as requested.

Check the logbook for the occurrence and source of a not-ready condition. Restart the analysis where required.

2 A not-ready condition was present during a sequence or multiple-injection run for a period longer than the timeout threshold.

Check the logbook for the occurrence and source of a not-ready condition. Restart the analysis where required.


1 Leak detected in another module with a CAN connection to the system.

Fix the leak in the external instrument before restarting the module.

2 Leak detected in an external instrument with a remote connection to the system.

Fix the leak in the external instrument before restarting the module.

3 Shut-down in an external instrument with a remote connection to the system.

Check external instruments for a shut-down condition.

4 The degasser failed to generate sufficient vacuum for solvent degassing.

Check the vacuum degasser for an error condition. Refer to the Service Manual for the degasser or the 1260 pump that has the degasser built-in.


Error Information 7General Error Messages

Remote Timeout

Error ID: 0070

A not- ready condition is still present on the remote input. When an analysis is started, the system expects all not- ready conditions (for example, a not- ready condition during detector balance) to switch to run conditions within one minute of starting the analysis. If a not- ready condition is still present on the remote line after one minute the error message is generated.

Lost CAN Partner

Error ID: 0071

During an analysis, the internal synchronization or communication between one or more of the modules in the system has failed.

The system processors continually monitor the system configuration. If one or more of the modules is no longer recognized as being connected to the system, the error message is generated.


1 Not-ready condition in one of the instruments connected to the remote line.

Ensure the instrument showing the not-ready condition is installed correctly, and is set up correctly for analysis.

2 Defective remote cable. Exchange the remote cable.

3 Defective components in the instrument showing the not-ready condition.

Check the instrument for defects (refer to the instrument’s documentation).


1 CAN cable disconnected. • Ensure all the CAN cables are connected correctly.

• Ensure all CAN cables are installed correctly.

2 Defective CAN cable. Exchange the CAN cable.

3 Defective main board in another module. Switch off the system. Restart the system, and determine which module or modules are not recognized by the system.



Leak

Error ID: 0064

A leak was detected in the module.

The signals from the two temperature sensors (leak sensor and board- mounted temperature- compensation sensor) are used by the leak algorithm to determine whether a leak is present. When a leak occurs, the leak sensor is cooled by the solvent. This changes the resistance of the leak sensor which is sensed by the leak- sensor circuit on the main board.

Leak Sensor Open

Error ID: 0083

The leak sensor in the module has failed (open circuit).

The current through the leak sensor is dependent on temperature. A leak is detected when solvent cools the leak sensor, causing the leak- sensor current to change within defined limits. If the current falls outside the lower limit, the error message is generated.


1 Loose fittings. Ensure all fittings are tight.

2 Broken capillary. Exchange defective capillaries.


1 Leak sensor not connected to the main board.

Please contact your Agilent service representative.

2 Defective leak sensor. Please contact your Agilent service representative.

3 Leak sensor incorrectly routed, being pinched by a metal component.



Error Information 7General Error Messages

Leak Sensor Short

Error ID: 0082

The leak sensor in the module has failed (short circuit).

The current through the leak sensor is dependent on temperature. A leak is detected when solvent cools the leak sensor, causing the leak sensor current to change within defined limits. If the current increases above the upper limit, the error message is generated.

Compensation Sensor Open

Error ID: 0081

The ambient- compensation sensor (NTC) on the main board in the module has failed (open circuit).

The resistance across the temperature compensation sensor (NTC) on the main board is dependent on ambient temperature. The change in resistance is used by the leak circuit to compensate for ambient temperature changes. If the resistance across the sensor increases above the upper limit, the error message is generated.


1 Defective leak sensor. Please contact your Agilent service representative.


1 Defective main board. Please contact your Agilent service representative.



Compensation Sensor Short

Error ID: 0080

The ambient- compensation sensor (NTC) on the main board in the module has failed (open circuit).

The resistance across the temperature compensation sensor (NTC) on the main board is dependent on ambient temperature. The change in resistance is used by the leak circuit to compensate for ambient temperature changes. If the resistance across the sensor falls below the lower limit, the error message is generated.

Fan Failed

Error ID: 0068

The cooling fan in the module has failed.

The hall sensor on the fan shaft is used by the main board to monitor the fan speed. If the fan speed falls below a certain limit for a certain length of time, the error message is generated.

Depending on the module, assemblies (e.g. the lamp in the detector) are turned off to assure that the module does not overheat inside.




1 Fan cable disconnected. Please contact your Agilent service representative.

2 Defective fan. Please contact your Agilent service representative.



Error Information 7Detector Error Messages

Detector Error Messages

Lamp Cover Open

Error ID: 6622, 6731

The lamp cover in the optical compartment has been removed. The lamp cannot be turned on while this message is on.

FLF Board not found

Error ID: 6620, 6730

The FLF board could not be found by the main board (FLM). This message comes together with some other message generated on the FLF board (e.g. Leak, ...).


1 Lamp cover removed. Please contact your Agilent service representative.


1 FLF board not connected to the FLM board. Please contact your Agilent service representative.

2 Defective FLF board. Please contact your Agilent service representative.

3 Defective FLM board. Please contact your Agilent service representative.


7 Error InformationDetector Error Messages

ADC Not Calibrated

Error ID: 6621, 6732

The analog- to- digital converter located on the FLF board cannot calibrate.

A/D Overflow

Error ID: 6618, 6619

This message is not implemented in firmware revision A.03.66 and below.

It indicates an overload situation of the A/D converter (sample signal). The user- interface will show a not- ready condition for the FLD and an info event is written into the logbook. If the message comes up during a run, it includes the time of occurrence and when it disappears.

1200 FLD 1 A/D overflow (RT is 0.32 min) 16:33:24 02/11/99

1200 FLD 1 A/D overflow finished (RT is 0.67 min)16:33:46 02/11/99

If this condition is present prior to a run, the not- ready will prevent the system to start the run/sequence.

With firmware revision A.06.11 and above, the A/D overflow leads into a flat peak in the chromatogram. For details see “Visualization of ADC Limits” on page 129.


1 Defective ADC or other FLF electronics. Please contact your Agilent service representative.


1 PMT setting to high. Reduce PMT gain.

2 Wavelength setting wrong. Change wavelength setting.



Flash Lamp Current Overflow

Error ID: 6704

The lamp current of the xenon flash lamp is monitored constantly. If the current gets too high, an error is generated and the lamp is turned OFF.

No light at reference diode despite lamp is on

Error ID: 6721

• Revision A/B/C Front End Board (FLF):

There is no feedback mechanism that checks whether the lamp is ON! If no peaks are shown in the chromatogram, the user- interface shows the module still in Ready. Perform a "Lamp Intensity Test" (see “Lamp Intensity Test” on page 160) first. If flat use below steps.

• Revision D Front End Board (FLF):

The flashing of the xenon flash lamp is monitored constantly. If the Lamp has not flashed for more than 100 times in series, an error is generated and the lamp is turned OFF.


1 Short-circuit of trigger pack assembly or defective FLL board.


2 Short-circuit of flash lamp assembly. Please contact your Agilent service representative.


1 Defective Hardware. Please contact your Agilent service representative.



Flash Trigger Lost

Error ID: 6722

This message is displayed when the flash trigger is no longer generated.

Wavelength Calibration Failed

Error ID: 6703

This message may show up during a wavelength calibration.

If the expected deviation is larger than the specified wavelength accuracy, the message “Wavelength Calibration Failed” is displayed and the instrument stays in a Not Ready condition.


1 Firmware problem. Reboot the detector (power cycle).

2 Multi Mode Off Please contact your Agilent service representative.

3 Defective encoder. Please contact your Agilent service representative.


1 Flash lamp not ignited or position not correct.


2 Cell position not correct. Check the cell position.

3 Solvent in the cell not clean or air bubble in the cell.

Flush the flow cell.

4 monochromator assembly position not correct (after replacement).




Wavelength Calibration Lost

Error ID: 6691

After exchanging the monochromator assemblies, the calibration factors should be reset to defaults values (a new FLM board comes with default values). In this case “Wavelength Calibration Lost” is displayed and the instrument stays in a Not Ready condition.

Flow Cell Removed

Error ID: 6616, 6702, 6760

The detector has an automatic cell recognition system. When the flow cell is removed, the lamp is turned off and a NOT READY condition exists. If the flow cell is removed during an analysis, a SHUT DOWN is generated.


1 Reset of monochromator settings after exchange.

Perform a wavelength calibration.

2 Replacement of FLM board. Perform a wavelength calibration.


1 Flow cell has been removed during analysis. Insert flow cell and turn on the lamp.



Motor Errors

If motor errors are displayed, please contact your Agilent service representative.

NOTE Monochromator motor errors may show up during the initialization or during operation of the detector.There are individual messages for either the excitation or the emission side.If an error occurs, do a lamp ignition. This will clear the error and a re-initialization of the motors is performed.



8Test Functions

Introduction 158

Diagram of Light Path 159

Lamp Intensity Test 160

Lamp Intensity History 161

Raman ASTM Signal-to-Noise Test 162

Using the Agilent Lab Advisor 165

Interpretation of the Results 165

Using the Built-in Test Chromatogram 166

Procedure Using the Agilent Lab Advisor 166

Wavelength Verification and Calibration 168

Wavelength Accuracy Test 171

Using the Agilent Lab Advisor 171

Interpretation of the Results 173

Using the Agilent ChemStation (Manually) 174

Wavelength Calibration Procedure 177

This chapter describes the detector’s built in test functions.


8 Test FunctionsIntroduction

Introduction

All tests are described based on the Agilent Lab Advisor Software B.02.03.

Other user interfaces may not provide any test or just a few.

For details on the use of the interface refer to the interface documentation.

Table 22 Interfaces and available test functions

Interface Comment Available Function

Agilent Instrument Utilities Maintenance tests are available

• Intensity• WL Calibration

Agilent Lab Advisor All tests are available • Intensity• ASTM Drift and Noise• Dark Current• D/A Converter• WL Accuracy• WL Calibration• Test Chromatogram (Tools)• Spectra Scan (Tools)• Module Infos (Tools)• Diagnostic (Tools)

Agilent ChemStation Some tests may be availableAdding of temperature

• Some of Lab Advisor tests

Agilent Instant Pilot Some tests are available • Intensity• WL Calibration• Spectra Scan (Tools)• Module Infos (Tools)• Diagnostic


Test Functions 8Diagram of Light Path

Diagram of Light Path

The light path is shown in Figure 56 on page 159.

Figure 56 Schematic of the Light Path


8 Test FunctionsLamp Intensity Test

Lamp Intensity Test

The intensity test scans an intensity spectrum via the reference diode (200 - 1200 nm in 1 nm steps) and stores it in a diagnosis buffer. The scan is displayed in a graphic window. There is no further evaluation of the test.

Results of this test are stored as lamp history (date code, intensity).

Figure 57 Lamp Intensity Test (Agilent Lab Advisor)

UV degradation, especially below 250 nm is significantly higher compared to visible wavelength range. Generally the "LAMP ON during run" setting or using "economy mode" will increase lamp life by a magnitude.

NOTE The profile can vary from instrument to instrument. It is dependig on the age of the lamp and the content of the flow cell (use fresh water).


Test Functions 8Lamp Intensity Test

Lamp Intensity History

Results of the lamp intensity test (if the last one is older than one week) are stored as lamp history (date code, intensity of four different wavelengths, 250 nm, 350 nm, 450 and 600 nm) in a buffer. The data/plot can be retrieved via the diagnostics and provides intensity data over a length of time.

Figure 58 Lamp Intensity History (Agilent Lab Advisor under Module Info)


8 Test FunctionsRaman ASTM Signal-to-Noise Test

Raman ASTM Signal-to-Noise Test

This test verifies the Raman ASTM signal- to- noise for the G1321 FLD detectors.

Figure 59 Raman ASTM Signal-to-Noise Test (Lab Advisor)

Depending on the version of the detector the specification has changed.

Conditions: Standard flow cell (G1321- 60005, G5615- 60005), flow of 0.25 mL/min of water.

Table 23 Raman ASTM Signal-to-Noise Specification

Instrument SNR Specification Raman / Dark

SNR Specification Dual WL

Comment

G1321C (1260) 500 / 3000 FLF board revision D and above

G1321B (1260) 500 / 3000 300 FLF board revision D and above

G1321A (1200) 500 300 FLF board revision D and above

G1321A (1100) 400 FLF board revision B and above

G1321A (1100) 200 FLF board revision A

NOTE The Dark and Dual WL values are just additional specifications. Only the Raman value is used for the standard instrument checkout.


Test Functions 8Raman ASTM Signal-to-Noise Test

NOTE The specification single wavelength at signal can be measured with the Agilent Lab Advisor. All others (not used for standard checkout) have to be set up manually with the information from Table 26 on page 163 and Table 27 on page 164.

Table 24 Raman Signal-to-Noise Test Conditions

Duration approximately 23 minutes

Standard Flow Cell G1321-60005, G5615-60005

Solvent LC grade water, degassed

Flow rate 0.25 mL/min

Specification (single wavelength at signal) >500 (according to settings in Table 25 on page 163)

Specification (single wavelength at background)

>3000 (according to settings in Table 26 on page 163)

Specification (dual wavelength) >300 (according to settings in Table 27 on page 164)

Table 25 Settings for Single Wavelength Specifications (at signal)

Time EX EM PMT Baseline

0 350 397 12 Free

20.30 350 450 12 Free

Table 26 Settings for Single Wavelength Specifications (at background)

Time EX EM PMT Baseline

0 350 450 14 Free

20.30 350 397 14 Free


8 Test FunctionsRaman ASTM Signal-to-Noise Test

)

Formulas for the Raman ASTM S/N value (see Figure 60 on page 164 for details):

Figure 60 Raman ASTM signal/noise calculation

Table 27 Settings for Dual Wavelength Specifications (Multi-EM Scan)

Time EX EM_A

EM_B

Spectra

From To Step

PMT

Baseline Fit Spectra

00.00

350 397 450 None 280 450 10 12 Free OFF

20.30

350 450 450 None 280 450 10 12 Free OFF

397)em 350,(exn noise_rama450 em 350,(ex roundmean_backg 397)em 350,(ex mean_ramanSNR_Raman

====−===

450) em 350,(ex groundnoise_back450) em 350,(ex roundmean_backg 397)em 350,(ex mean_ramanSNR_Dark

====−===


Test Functions 8Raman ASTM Signal-to-Noise Test

Using the Agilent Lab Advisor

1 Set up the HPLC system and the Lab Advisor.

2 Flush the flow cell with clean bidistilled water.

3 Start the test in the Lab Advisor.

Figure 61 Raman ASTM Signal-to-Noise Test (Agilent Lab Advisor)

In case of failing this test (as shown above) see “Interpretation of the Results” on page 165.

Interpretation of the Results

If the test shows low Raman values, check for:

✔ correctly positioned flow cell,

✔ clean flow cell (flush with clean bidistilled water),

✔ no air bubble(s) (check via fluorescence scan or visual check of cell/cuvette),

✔ solvent inlet filter (may create air bubbles in flow cell).


8 Test FunctionsUsing the Built-in Test Chromatogram

Using the Built-in Test Chromatogram

This function is available from the Agilent ChemStation, Lab Advisor and Instant Pilot.

The built- in Test Chromatogram can be used to check the signal path from the detector to the data system and the data analysis or via the analog output to the integrator or data system. The chromatogram is continuously repeated until a stop is executed either by means of a stop time or manually.

Procedure Using the Agilent Lab Advisor

This procedure works for all Agilent 1200 Infinity detectors (DAD, MWD, VWD, FLD and RID). The example figure is from the RID detector.

1 Assure that the default LC method is loaded via the control software.

2 Start the Agilent Lab Advisor software (B.01.03 SP4 or later) and open the detector's Tools selection.

3 Open the test chromatogram screen

4 Turn the Test Chromatogram on.

5 Change to the detector's Module Service Center and add the detector signal to the Signal Plot window.

NOTE The peak height is always the same but the area and the retention time depend on the set peakwidth, see example below.


Test Functions 8Using the Built-in Test Chromatogram

6 To start a test chromatogram enter in the command line: STRT

Figure 62 Test Chromatogram with Agilent Lab Advisor

7 To stop the test chromatogram enter in the command line: STOP

NOTE The test chromatogram is switched off automatically at the end of a run.


8 Test FunctionsWavelength Verification and Calibration

Wavelength Verification and Calibration

The wavelength calibration is based on a Glycogen solution, which acts as a strong elastic light scatterer (refer to ASTM Test Method E388- 72- 1993 “Spectral Bandwidth and Wavelength Accuracy of Fluorescence Spectrometers”). The Glycogen solution is introduced into the flow cell and then the built- in wavelength calibration functionality is used.

The algorithm is based on evaluating different grating orders and calculating the wavelength scales of both, excitation and emission monochromator, by applying the fundamental grating equation.

( X ) only required, if deviation is too large.

NOTE A complete wavelength calibration is not always required. In most cases a quick wavelength accuracy verification is sufficient enough, see Table 28 on page 168.

Table 28 Reasons for doing a Verification or Calibration

Verification WL calibration

interest X

GLP compliance X

cell change X ( X )

lamp change X ( X )

monochromator change X

main board change X

optical unit change X

NOTE Prior to a wavelength calibration, a wavelength accuracy verification should be performed, see “Wavelength Accuracy Test” on page 171. If the deviation is more than ±3 nm, the wavelength calibration should be done as described in “Wavelength Calibration Procedure” on page 177.


Test Functions 8Wavelength Verification and Calibration

Table 29 on page 170 shows the steps performed during the wavelength calibration.

The excitation grating and the emission grating are calibrated using Rayleigh stray light from the flow cell or cuvette measured with the photomultiplier tube.

Figure 63 Wavelength Calibration (Agilent Lab Advisor)

NOTE The duration of the wavelength calibration is about 15 minutes plus setup time for the calibration sample and system. Depending on the maximum intensity found during this scan, the PMT gain will be changed automatically and requires an additional 1 minute per scan.


8 Test FunctionsWavelength Verification and Calibration

If you encounter calibration problems:

1 Check for air bubbles in the flow cell.

2 Flush the flow cell with isopropanol.

3 Change the water.

Table 29 Wavelength Calibration Steps

Step Description Duration

1 Preparation max 30 s

2 Excitation rotation scan, full circle 60 s

3 Excitation rotation scan, high resolution 44 s

4 Excitation position scan, low resolution 55 s variable

5 Excitation position scan, high resolution 260 s variable

6.n Emission rotation scan, full circle (# of scans depends on the required PMT gain, 1 minute per scan)

61 s variable

6.n Em rotation scan, full circle" (instrument profile) 9 s




7 Emission rotation scan, high resolution, part I 44 s

8 Emission rotation scan, high resolution, part II 44 s

9 Emission position scan, low resolution 50 s variable

10 Emission position scan, high resolution 250 s variable

NOTE Variable times means that they could be a little bit longer.

When the lamp is off, the calibration process will stop within the first two steps with “ Wavelength Calibration Failed” , see “Wavelength Calibration Failed” on page 154.


Test Functions 8Wavelength Accuracy Test

Wavelength Accuracy Test

Using the Agilent Lab Advisor

1 Set up the HPLC system and the Agilent Lab Advisor.

2 Flush the flow cell with clean bidistilled water.

3 Turn on the FLD lamp.

4 Run the Wavelength Accuracy Test.

5 The FLD will change into the multi- excitation mode with emission wavelength at 397 nm and scan in the range of the expected maximum of 350 nm±20 nm.

As result, the maxima should be found at 350 nm ±3 nm, see Figure 64 on page 171.

The FLD will change into the multi- emission mode with excitation wavelength at 350 nm and scan in the range of the expected maximum of 397 nm±20 nm.

As result, the maxima should be found at 397 nm ±3 nm, see Figure 64 on page 171.

Figure 64 Excitation and Emission Spectrum (expected results)


8 Test FunctionsWavelength Accuracy Test

Figure 65 Wavelength Accuracy Test with Lab Advisor

If the test fails observe the maxima of the EX or EM side under the Signals tab.

Figure 66 Example of good EX maxima

If the plots do not have a maximum around EX=397 nm and EX=350 nm (±3 nm) the test fails, see figure below. Refer to “Interpretation of the Results” on page 173.

NOTE If the plots do not have a maximum around EM=397 nm and EX=350 nm (±3 nm) the test fails. Refer to “Interpretation of the Results” on page 173.



Figure 67 Example of bad EX/EM maxima (no maximum found)

Interpretation of the Results

If the test fails, check for:

✔ correctly positioned flow cell,

✔ clean flow cell (flush with isopropanol and clean bidistilled water),

✔ no air bubble(s) (check via fluorescence scan or visual check of cell/cuvette),

✔ solvent inlet filter (may create air bubbles in flow cell).

✔ check optical path for contamination (service)

✔ check alignment of lamp / trigger pack assembly (service)

✔ perform a Wavelength Calibration



Using the Agilent ChemStation (Manually)

1 Create the methods WLEMTEST and WLEXTEST as listed Table 30 on page 174.

Table 30 Method Settings

Setting Check of EM WL 397 nmWLEMTEST

Check of EX WL 350 nmWLEXTEST

Peak Width >0.2 min (4 s, standard) >0.2 min (4 s, standard)

Fit Spectral Range OFF OFF

PMT Gain 12 12

Flash Lamp ON ON

Spectrum Range EM 367 - 417 nm step 1 nm EX 330 - 380 nm step 1 nm

Store Spectra All w/o signal All w/o signal

EX Wavelength 350 nm, ON 350 nm, OFF

EM Wavelength 397 nm, OFF 397 nm, ON

Multi WL Settings Multi EM Multi EX



Figure 68 Special Setpoints Settings

Figure 69 Settings for EM / EX Scan



2 Load the method WLEXTEST. The FLD will change into the multi- emission mode and scan in the range of the expected maximum of 397 nm ±20 nm.

3 Start the pump and flush the cell with water for a few minutes to assure a clean flow cell. Flow rate should be 0.5 to 1 ml/min and the baseline stable.

4 Open the Online Spectra plot and observe the maximum as shown in Figure 64 on page 171 (left).

5 Load the method WLEMTEST. The FLD will change into the multi- excitation mode and scan in the range of the expected maximum of 350 nm ±20 nm.

6 Open the Online Spectra plot and observe the maximum as shown in Figure 64 on page 171 (right).

NOTE You may remove the flow cell and check for air bubbles. After re-inserting the cell, turn the lamp ON.


Test Functions 8Wavelength Calibration Procedure

Wavelength Calibration Procedure

1 Preparation of the Glycogen Calibration Sample.

a To prepare 10 ml of the calibration solution you have to use 10 mg of the Glycogen sample (a tolerance of ±20% is not critical).

b Fill the prepared amount into a suitable bottle/vial.

c Fill 10 ml of distilled water into the vial and shake.

d Wait 5 minutes and shake again. After 10 minutes the solution is ready.

2 Preparation of the Flow Cell.

a Flush the flow cell with water.

b Remove the inlet capillary from the flow cell.

c Take the syringe and fix the needle to the syringe adapter.

d Suck about 1.0 ml of the calibration sample into the syringe.

e Keep the syringe in a horizontal position.

f Remove the needle.

When If application requires, or see Table 29 on page 170.


Laboratory balance


5063-6597 Calibration Sample, Glycogen

9301-1446 Syringe

9301-0407 Needle

5190-5111 Syringe filter, 0.45 µm, 100/pk

0100-1516 Fitting male PEEK, 2/pk


8 Test FunctionsWavelength Calibration Procedure

g Add the filter to the syringe and fit the needle to filter.

Figure 70 Syringe with Sample Filter

h Lift the needle tip and carefully eject approximately 0.5 ml to remove air out of the syringe and to flush the needle.

i Add the PEEK fitting to the needle tip and fix both at the flow cell inlet.

j Slowly inject about 0.2 ml and wait for about 10 seconds to inject another 0.1 ml. This will assure that the cell is filled properly.

3 Wavelength Calibration.

a From the user interface start the FLD wavelength calibration (see Figure 73 on page 180).

• Agilent Lab Advisor: Calibrations

• Agilent ChemStation: Diagnosis > Maintenance > FLD Calibration

• Instant Pilot (G4208A): Maintenance > FLD > Calibration

NOTE Do not inject the calibration sample without the sample filter.

NOTE If the wavelength calibration process fails, refer to “Wavelength Calibration Failed” on page 154.



b If a deviation is displayed, press Yes (Lab Advisor) to adjust to new values or Adjust and OK (ChemStation, see next page). The history table will be updated.

Figure 71 Wavelength Calibration (Agilent Lab Advisor)



Figure 72 Calibration History (Agilent Lab Advisor, under Module Info)

Figure 73 Wavelength Calibration (Agilent ChemStation)

NOTE To look at the history table (ChemStation) start a wavelength calibration and abort immediately. No changes are made to the calibration at this time.



4 Verification using “Wavelength Accuracy Test” on page 171.

a Refit the capillary to the flow cell.

b Follow the procedure “Wavelength Accuracy Test” on page 171.

NOTE Rinse the flow cell with pure water at a minimum of 1.5 ml/min to get rid of the Glycogen from the cell and the capillaries. When organic solvent is sequentially applied (without rinsing), a blockage of capillaries may occur.



9Maintenance

Introduction to Maintenance 184

Warnings and Cautions 185

Overview of Maintenance 187

Cleaning the Module 188

Exchanging a Flow Cell 189

How to use the Cuvette 193

Flow Cell Flushing 194

Correcting Leaks 195

Replacing Leak Handling System Parts 196

Replacing the Interface Board 197

Replacing Module Firmware 198

Tests and Calibrations 199

This chapter provides general information on maintenance of the detector.


9 MaintenanceIntroduction to Maintenance

Introduction to Maintenance

The module is designed for easy maintenance. Maintenance can be done from the front with module in place in the system stack.

NOTE There are no serviceable parts inside.

Do not open the module.


Maintenance 9Warnings and Cautions

Warnings and Cautions






WARNING Eye damage by detector light

Eye damage may result from directly viewing the UV-light produced by the lamp of the optical system used in this product.

➔ Always turn the lamp of the optical system off before removing it.

WARNING Electrical shock

Repair work at the module can lead to personal injuries, e.g. shock hazard, when the cover is opened.

➔ Do not remove the cover of the module.

➔ Only certified persons are authorized to carry out repairs inside the module.


9 MaintenanceWarnings and Cautions

WARNING Personal injury or damage to the product

Agilent is not responsible for any damages caused, in whole or in part, by improper use of the products, unauthorized alterations, adjustments or modifications to the products, failure to comply with procedures in Agilent product user guides, or use of the products in violation of applicable laws, rules or regulations.

➔ Use your Agilent products only in the manner described in the Agilent product user guides.

CAUTION Safety standards for external equipment

➔ If you connect external equipment to the instrument, make sure that you only use accessory units tested and approved according to the safety standards appropriate for the type of external equipment.


Maintenance 9Overview of Maintenance

Overview of Maintenance

The following pages describe maintenance (simple repairs) of the detector that can be carried out without opening the main cover.

Table 31 Simple Repairs

Procedure Typical Frequency Notes

Flow cell exchange If application requires a different flow cell type or if defective.

Complete AssemblyA wavelength calibration check should be performed after replacement.

If the flow cell is removed and inserted, then a quick calibration check is performed.If this fails, you must do a wavelength recalibration, see “Wavelength Verification and Calibration” on page 168.

Flow cell flushing If flow cell is contaminated.

Leak sensor drying If leak has occurred. Check for leaks.

Leak handling System replacement

If broken or corroded. Check for leaks.


9 MaintenanceCleaning the Module

Cleaning the Module

To keep the module case clean, use a soft cloth slightly dampened with water, or a solution of water and mild detergent.

WARNING Liquid dripping into the electronic compartment of your module can cause shock hazard and damage the module

➔ Do not use an excessively damp cloth during cleaning.

➔ Drain all solvent lines before opening any connections in the flow path.


Maintenance 9Exchanging a Flow Cell

Exchanging a Flow Cell

For bio-inert modules use bio-inert parts only!

When If an application needs a different type of flow cell or the flow cell is defective (leaky).


Wrench, 1/4 inchfor capillary connections

OR 5043-0915 Fitting mounting toolfor bio-inert capillaries


1 G1321-60005 Flow cell, 8 µL, 20 bar (pH 1 – 9.5 )

1 G1321-60015 Flow cell, 4 µL, 20 bar (pH 1 – 9.5 )

1 G5615-60005 Bio-inert flow cell, 8 µL, 20 bar (pH 1–12) includes Capillary Kit Flow Cells BIO (p/n G5615-68755)

1 G1321-60007 FLD Cuvette Kit, 8 µL, 20 bar

Preparations Turn off the flow.

CAUTION Sample degradation and contamination of the instrument

Metal parts in the flow path can interact with the bio-molecules in the sample leading to sample degradation and contamination.

➔ For bio-inert applications, always use dedicated bio-inert parts, which can be identified by the bio-inert symbol or other markers described in this manual.

➔ Do not mix bio-inert and non-inert modules or parts in a bio-inert system.

NOTE DO NOT install the inlet capillary to the outlet connection of the flow cell. This will result in poor performance.

NOTE In case the flow cell is not used for some time (stored), then flush the flow cell with iso-propanol and close the cell with Plug-Screw - Fitting (0100-1259).


9 MaintenanceExchanging a Flow Cell

1 Press the release buttons and remove the front cover for access to the flow cell area.

2 Disconnect the capillaries from the flow cell.


Maintenance 9Exchanging a Flow Cell

3 Unscrew the thumb screws and pull the flow cell out of the compartment.

NOTEThe label attached to the flow cell provides information on part number, cell volume and maximum pressure. The cell type will be automatically detected.

There are no parts that can be replaced on the flow cell. If defective (leaky) the flow cell has to be replaced completely.

4 Insert the flow cell and tighten the thumb screws. Reconnect the capillaries to the flow cell. DO NOT install the inlet capillary to the outlet connection of the flow cell. This will result in poor performance or damage.

NOTEIf an additional detector is added to the system, the fluorescence detector should be the last detector in the flow path except for evaporative detectors, like LC-MSD. Otherwise the back pressure generated by the other detector may overload the flow cell and will lead to a defective cell (maximum pressure is 20 bar (2 MPa)).

Always use the outlet capillary set supplied with the accessory kit.

NOTETo check for leaks, establish a flow and observe the flow cell (outside of the cell compartment) and all capillary connections.


9 MaintenanceExchanging a Flow Cell

5 Replace the front cover.

NOTEPerform a wavelength verification to check the correct positioning of the flow cell, as described in chapter “Wavelength Verification and Calibration” on page 168.


Maintenance 9How to use the Cuvette

How to use the Cuvette

The cuvette is used for offline measurements (no flow system required) and is basically a standard flow cell with a few changes:

• wide bore capillary connections for easier injections with a syringe

• identification lever for cell auto- recognition system.

1 Install the cuvette instead of the standard flow cell.

2 Connect the waste tubing to the outlet of the cuvette.

3 Use the syringe (see “Cuvette Kit” on page 203) to inject the compound.

4 Setup the parameters for the Fluorescence Scan (under Special Setpoints).

5 Select “Take Fluorescence Scan” on the user- interface to start the offline measurement.


9 MaintenanceFlow Cell Flushing

Flow Cell Flushing

1 Flush with bidistilled water.

2 Flush with nitric acid (65 %) using a glass syringe.

3 Leave this solution in the cell for about one hour.

4 Flush with bidistilled water.

When If flow cell is contaminated


Glass syringe

Adapter

Parts required # Description

1 Bidistilled water, nitric acid (65 %), tubings to waste

WARNING Dangerous concentration of nitric acid

The nitric acid flushing procedure is not an infallible remedy for a dirty cell. It is to be used as a last attempt to salvage the cell before cell replacement. Note that the cell is a consumable item.

➔ Give proper attention to safety.

NOTE Aqueous solvents in the flow cell can built up algae. Algae do fluoresce. Therefore do not leave aqueous solvents in the flow cell for longer periods. Add a small percentage of organic solvents (e.g. Acetonitrile or Methanol ~5 %).

NOTE Do not exceed the pressure limit of 20 bar (0.2 MPa).


Maintenance 9Correcting Leaks

Correcting Leaks

1 Remove the front cover.

2 Use tissue to dry the leak sensor area and the leak pan.

3 Observe the capillary connections and the flow cell area for leaks and correct, if required.


Figure 74 Observing for Leaks

When If a leakage has occurred in the flow cell area or at the capillary connections


Tissue

Wrench, 1/4 inchfor capillary connections

5043-0915 Fitting mounting toolfor bio-inert capillaries


9 MaintenanceReplacing Leak Handling System Parts

Replacing Leak Handling System Parts

1 Remove the front cover.

2 Pull the leak funnel out of the leak funnel holder.

3 Pull out the leak funnel with the tubing.

4 Insert the leak funnel with the tubing in its position.

5 Insert the leak funnel into the leak funnel holder.


Figure 75 Replacing Leak Handling System Parts

When If the parts are corroded or broken


1 5041-8388 Leak funnel

1 5041-8389 Leak funnel holder

1 5042-9974 Leak tubing (1.5 m, 120 mm required)


Maintenance 9Replacing the Interface Board

Replacing the Interface Board

1 To replace the interface board unscrew the two screws, remove the board, slide in the new interface board and fix it with the board’s screws.

Figure 76 Location of the Interface Board

When For all repairs inside the detector or for installation of the board


1 G1351-68701 Interface board (BCD) with external contacts and BCD outputs

1 G1369B or G1369-60002

Interface board (LAN)

OR 1 G1369C or G1369-60012



9 MaintenanceReplacing Module Firmware

Replacing Module Firmware

To upgrade/downgrade the module’s firmware carry out the following steps:

1 Download the required module firmware, the latest LAN/RS- 232 FW Update Tool and the documentation from the Agilent web.

• http://www.chem.agilent.com/_layouts/agilent/downloadFirmware.aspx?whid=69761

2 For loading the firmware into the module follow the instructions in the documentation.

Module Specific Information

There is no specific information for this module.

When The installation of newer firmware might be necessary• if a newer version solves problems of older versions or• to keep all systems on the same (validated) revision.

The installation of older firmware might be necessary• to keep all systems on the same (validated) revision or• if a new module with newer firmware is added to a system or• if third party control software requires a special version.


LAN/RS-232 Firmware Update Tool

OR Agilent Lab Advisor software

OR Instant Pilot G4208A (only if supported by module)

Parts required # Description

1 Firmware, tools and documentation from Agilent web site

Preparations Read update documentation provided with the Firmware Update Tool.


Maintenance 9Tests and Calibrations

Tests and Calibrations

The following tests are required after maintenance of lamps and flow cells:

• “Lamp Intensity Test” on page 160.

• “Wavelength Verification and Calibration” on page 168


9 MaintenanceTests and Calibrations



10Parts for Maintenance

Overview of Maintenance Parts 202

Cuvette Kit 203

Accessory Kit 204

This chapter provides information on parts for maintenance.


10 Parts for MaintenanceOverview of Maintenance Parts

Overview of Maintenance Parts

Parts for wavelength calibration, see “Standard Accessory Kit” on page 204.

p/n Description

G1321-60005 Flow cell, 8 µL, 20 bar (pH 1 – 9.5 )

OR G1321-60015 Flow cell, 4 µL, 20 bar (pH 1 – 9.5 ) requires a 0.12 mm i.d. capillary (e.g. p/n G1316-87318, 300 mm long), part of Capillary kit for 0.12 mm id (p/n G1316-68716)

OR G5615-60005 Bio-inert flow cell, 8 µL, 20 bar (pH 1–12) includes Capillary Kit Flow Cells BIO (p/n G5615-68755)

G5615-68755 Capillary Kit Flow Cells BIO includes Capillary PK 0.18 mm x 1.5 m and PEEK Fittings 10/PK (p/n 5063-6591)

G1321-60007 FLD Cuvette Kit, 8 µL, 20 bar

9301-0407 Needle

9301-1446 Syringe

5067-4691 Front Panel DAD/VWD/FLD (1260/1290)

5041-8388 Leak funnel

5041-8389 Leak funnel

5041-8387 Tube clip

5062-2463 Corrugated tubing, PP, 6.5 mm id, 5 m

5062-2462 Tube PTFE 0.8 mm x 2 m, re-order 5 m

5181-1516 CAN cable, Agilent module to module, 0.5 m

5181-1519 CAN cable, Agilent module to module, 1 m

G1369B or G1369-60002


5023-0203 Cross-over network cable, shielded, 3 m (for point to point connection)

5023-0202 Twisted pair network cable, shielded, 7 m (for point to point connection)

01046-60105 Agilent module to general purpose (Analog)

G1351-68701 Interface board (BCD) with external contacts and BCD outputs


Parts for Maintenance 10Cuvette Kit

Cuvette Kit

p/n Description

G1321-60007 FLD Cuvette Kit, 8 µL, 20 bar includes:

5062-2462 Tube PTFE 0.8 mm x 2 m, re-order 5 m

79814-22406 ST Fitting

0100-0043 ST front ferrule

0100-0044 ST back ferrule

0100-1516 Fitting male PEEK, 2/pk

9301-0407 Needle

9301-1446 Syringe


10 Parts for MaintenanceAccessory Kit

Accessory Kit

Standard Accessory Kit

Accessory kit (G1321- 68755) contains some accessories and tools needed for the installation and repair/calibration of the detector.

Figure 77 Waste Tubing Parts

Item p/n Description

1 5062-2462 Tube PTFE 0.8 mm x 2 m, re-order 5 m

2 0100-1516 Fitting male PEEK, 2/pk

3 G1315-87311 Capillary ST 0.17 mm x 380 mm S/SColumn to detector (includes ST ferrule front, ST ferrule back and ST fitting)

4 0100-0043 ST front ferrule

5 0100-0044 ST back ferrule

6 79814-22406 ST Fitting


Parts for Maintenance 10Accessory Kit

Figure 78 Inlet Capillary (Column-Detector) Parts

Capillary Kit Flow Cells BIO

Capillary Kit Flow Cells BIO includes Capillary PK 0.18 mm x 1.5 m and PEEK Fittings 10/PK (p/n 5063- 6591) (G5615- 68755) includes:

p/n Description

0890-1763 Capillary PK 0.18 mm x 1.5 m

5063-6591 PEEK Fittings 10/PK


10 Parts for MaintenanceAccessory Kit



11Identifying Cables

Cable Overview 208

Analog Cables 210

Remote Cables 212

BCD Cables 215

CAN/LAN Cables 217

External Contact Cable 218

Agilent Module to PC 219

This chapter provides information on cables used with the Agilent 1200 Infinity Series modules.


11 Identifying CablesCable Overview

Cable Overview

Analog cables

Remote cables

BCD cables

NOTE Never use cables other than the ones supplied by Agilent Technologies to ensure proper functionality and compliance with safety or EMC regulations.

p/n Description

35900-60750 Agilent module to 3394/6 integrators

35900-60750 Agilent 35900A A/D converter

01046-60105 Analog cable (BNC to general purpose, spade lugs)

p/n Description

03394-60600 Agilent module to 3396A Series I integrators

3396 Series II / 3395A integrator, see details in section “Remote Cables” on page 212

03396-61010 Agilent module to 3396 Series III / 3395B integrators

5061-3378 Remote Cable

01046-60201 Agilent module to general purpose

p/n Description

03396-60560 Agilent module to 3396 integrators

G1351-81600 Agilent module to general purpose


Identifying Cables 11Cable Overview

CAN cables

LAN cables

RS-232 cables

p/n Description



p/n Description



p/n Description

G1530-60600 RS-232 cable, 2 m

RS232-61601 RS-232 cable, 2.5 mInstrument to PC, 9-to-9 pin (female). This cable has special pin-out, and is not compatible with connecting printers and plotters. It's also called "Null Modem Cable" with full handshaking where the wiring is made between pins 1-1, 2-3, 3-2, 4-6, 5-5, 6-4, 7-8, 8-7, 9-9.

5181-1561 RS-232 cable, 8 m


11 Identifying CablesAnalog Cables

Analog Cables

One end of these cables provides a BNC connector to be connected to Agilent modules. The other end depends on the instrument to which connection is being made.

Agilent Module to 3394/6 Integrators

Agilent Module to BNC Connector

p/n 35900-60750 Pin 3394/6 Pin Agilent module

Signal Name

1 Not connected

2 Shield Analog -

3 Center Analog +

p/n 8120-1840 Pin BNC Pin Agilent module

Signal Name

Shield Shield Analog -

Center Center Analog +


Identifying Cables 11Analog Cables

Agilent Module to General Purpose

p/n 01046-60105 Pin Pin Agilent module

Signal Name

1 Not connected

2 Black Analog -

3 Red Analog +


11 Identifying CablesRemote Cables

Remote Cables

One end of these cables provides a Agilent Technologies APG (Analytical Products Group) remote connector to be connected to Agilent modules. The other end depends on the instrument to be connected to.

Agilent Module to 3396A Integrators

Agilent Module to 3396 Series II / 3395A Integrators

Use the cable Agilent module to 3396A Series I integrators (03394- 60600) and cut pin #5 on the integrator side. Otherwise the integrator prints START; not ready.

p/n 03394-60600 Pin 3396A Pin Agilent module

Signal Name Active (TTL)

9 1 - White Digital ground

NC 2 - Brown Prepare run Low

3 3 - Gray Start Low

NC 4 - Blue Shut down Low

NC 5 - Pink Not connected

NC 6 - Yellow Power on High

5,14 7 - Red Ready High

1 8 - Green Stop Low

NC 9 - Black Start request Low

13, 15 Not connected


Identifying Cables 11Remote Cables

Agilent Module to 3396 Series III / 3395B Integrators

Agilent Module to Agilent 35900 A/D Converters

p/n 03396-61010 Pin 33XX Pin Agilent module


9 1 - White Digital ground

NC 2 - Brown Prepare run Low

3 3 - Gray Start Low

NC 4 - Blue Shut down Low

NC 5 - Pink Not connected

NC 6 - Yellow Power on High

14 7 - Red Ready High

4 8 - Green Stop Low

NC 9 - Black Start request Low

13, 15 Not connected

p/n 5061-3378 Pin 35900 A/D

Pin Agilent module


1 - White 1 - White Digital ground

2 - Brown 2 - Brown Prepare run Low

3 - Gray 3 - Gray Start Low

4 - Blue 4 - Blue Shut down Low

5 - Pink 5 - Pink Not connected

6 - Yellow 6 - Yellow Power on High

7 - Red 7 - Red Ready High

8 - Green 8 - Green Stop Low

9 - Black 9 - Black Start request Low


11 Identifying CablesRemote Cables


p/n 01046-60201 Wire Color Pin Agilent module


White 1 Digital ground

Brown 2 Prepare run Low

Gray 3 Start Low

Blue 4 Shut down Low

Pink 5 Not connected

Yellow 6 Power on High

Red 7 Ready High

Green 8 Stop Low

Black 9 Start request Low


Identifying Cables 11BCD Cables

BCD Cables

One end of these cables provides a 15- pin BCD connector to be connected to the Agilent modules. The other end depends on the instrument to be connected to


p/n G1351-81600 Wire Color Pin Agilent module

Signal Name BCD Digit

Green 1 BCD 5 20

Violet 2 BCD 7 80

Blue 3 BCD 6 40

Yellow 4 BCD 4 10

Black 5 BCD 0 1

Orange 6 BCD 3 8

Red 7 BCD 2 4

Brown 8 BCD 1 2

Gray 9 Digital ground Gray

Gray/pink 10 BCD 11 800

Red/blue 11 BCD 10 400

White/green 12 BCD 9 200

Brown/green 13 BCD 8 100

not connected 14

not connected 15 + 5 V Low


11 Identifying CablesBCD Cables

Agilent Module to 3396 Integrators

p/n 03396-60560 Pin 3396 Pin Agilent module

Signal Name BCD Digit

1 1 BCD 5 20

2 2 BCD 7 80

3 3 BCD 6 40

4 4 BCD 4 10

5 5 BCD0 1

6 6 BCD 3 8

7 7 BCD 2 4

8 8 BCD 1 2

9 9 Digital ground

NC 15 + 5 V Low


Identifying Cables 11CAN/LAN Cables

CAN/LAN Cables

Both ends of this cable provide a modular plug to be connected to Agilent modules CAN or LAN connectors.

CAN Cables

LAN Cables

p/n Description



p/n Description




11 Identifying CablesExternal Contact Cable

External Contact Cable

One end of this cable provides a 15- pin plug to be connected to Agilent modules interface board. The other end is for general purpose.

Agilent Module Interface Board to general purposes

p/n G1103-61611 Color Pin Agilent module

Signal Name

White 1 EXT 1

Brown 2 EXT 1

Green 3 EXT 2

Yellow 4 EXT 2

Grey 5 EXT 3

Pink 6 EXT 3

Blue 7 EXT 4

Red 8 EXT 4

Black 9 Not connected

Violet 10 Not connected

Grey/pink 11 Not connected

Red/blue 12 Not connected

White/green 13 Not connected

Brown/green 14 Not connected

White/yellow 15 Not connected

51015

1

116


Identifying Cables 11Agilent Module to PC

Agilent Module to PC

p/n Description

G1530-60600 RS-232 cable, 2 m

RS232-61601 RS-232 cable, 2.5 mInstrument to PC, 9-to-9 pin (female). This cable has special pin-out, and is not compatible with connecting printers and plotters. It's also called "Null Modem Cable" with full handshaking where the wiring is made between pins 1-1, 2-3, 3-2, 4-6, 5-5, 6-4, 7-8, 8-7, 9-9.

5181-1561 RS-232 cable, 8 m


11 Identifying CablesAgilent Module to PC



12Hardware Information

Firmware Description 222

Optional Interface Boards 225

Electrical Connections 229

Rear view of the module 230

Serial Number Information 231

Interfaces 232

Overview Interfaces 235

Setting the 8-bit Configuration Switch (without On-board LAN) 239

Communication Settings for RS-232C 240

Special Settings 242

Early Maintenance Feedback 243

Instrument Layout 244

This chapter describes the detector in more detail on hardware and electronics.


12 Hardware InformationFirmware Description

Firmware Description

The firmware of the instrument consists of two independent sections:

• a non- instrument specific section, called resident system

• an instrument specific section, called main system

Resident System

This resident section of the firmware is identical for all Agilent 1100/1200/1220/1260/1290 series modules. Its properties are:

• the complete communication capabilities (CAN, LAN and RS- 232C)

• memory management

• ability to update the firmware of the 'main system'

Main System

Its properties are:

• the complete communication capabilities (CAN, LAN and RS- 232C)

• memory management

• ability to update the firmware of the 'resident system'

In addition the main system comprises the instrument functions that are divided into common functions like

• run synchronization through APG remote,

• error handling,

• diagnostic functions,

• or module specific functions like

• internal events such as lamp control, filter movements,

• raw data collection and conversion to absorbance.

Firmware Updates

Firmware updates can be done using your user interface:

• PC and Firmware Update Tool with local files on the hard disk


Hardware Information 12Firmware Description

• Instant Pilot (G4208A) with files from a USB Flash Disk

• Agilent Lab Advisor software B.01.03 and above

The file naming conventions are:

PPPP_RVVV_XXX.dlb, where

PPPP is the product number, for example, 1315AB for the G1315A/B DAD,

R the firmware revision, for example, A for G1315B or B for the G1315C DAD,

VVV is the revision number, for example 102 is revision 1.02,

XXX is the build number of the firmware.

For instructions on firmware updates refer to section Replacing Firmware in chapter "Maintenance" or use the documentation provided with the Firmware Update Tools.

Figure 79 Firmware Update Mechanism

NOTE Update of main system can be done in the resident system only. Update of the resident system can be done in the main system only.

Main and resident firmware must be from the same set.


12 Hardware InformationFirmware Description

The firmware update tools, firmware and documentation are available from the Agilent web.

• http://www.chem.agilent.com/_layouts/agilent/downloadFirmware.aspx?whid=69761

NOTE Some modules are limited in downgrading due to their main board version or their initial firmware revision. For example, a G1315C DAD SL cannot be downgraded below firmware revision B.01.02 or to a A.xx.xx.

Some modules can be re-branded (e.g. G1314C to G1314B) to allow operation in specific control software environments. In this case the feature set of the target type are use and the feature set of the original are lost. After re-branding (e.g. from G1314B to G1314C), the original feature set is available again.

All these specific informations are described in the documentation provided with the firmware update tools.


Hardware Information 12Optional Interface Boards

Optional Interface Boards

BCD / External Contact Board

The Agilent 1200 Infinity Series modules have one optional board slot that allows to add an interface board to the modules. Some modules do not have this interface slot. Refer to “Interfaces” on page 232 for details.

Optional Interface Boards

The BCD board provides a BCD output for the bottle number of the Agilent 1200 Series autosampler and four external contacts. The external contact closure contacts are relay contacts. The maximum settings are: 30 V (AC/DC); 250 mA (fused).

p/n Description

G1351-68701 Interface board (BCD) with external contacts and BCD outputs

2110-0004 Fuse for BCD board, 250 mA


12 Hardware InformationOptional Interface Boards

There are general purpose cables available to connect the BCD output, see “BCD Cables” on page 215 and the external outputs, see “External Contact Cable” on page 218 to external devices.

Table 32 Detailed connector layout (1200)

Pin Signal name BCD digit

1 BCD 5 20

2 BCD 7 80

3 BCD 6 40

4 BCD 4 10

5 BCD 0 1

6 BCD 3 8

7 BCD 2 4

8 BCD 1 2

9 Digital ground

10 BCD 11 800

11 BCD 10 400

12 BCD 9 200

13 BCD 8 100

15 +5V Low


Hardware Information 12Optional Interface Boards

LAN Communication Interface Board

The Agilent modules have one optional board slot that allows to add an interface board to the modules. Some modules do not have this interface slot. Refer to “Interfaces” on page 232 for details.

The following cards can be used with the Agilent 1260 Infinity modules.

p/n Description

G1369B or G1369-60002


OR G1369C or G1369-60012


NOTE One board is required per Agilent 1260 Infinity instrument. It is recommended to add the LAN board to the detector with highest data rate.

NOTE For the configuration of the G1369 LAN Communication Interface card refer to its documentation.

Table 33 LAN Boards

Type Vendor Supported networks

Interface board (LAN) (G1369B or G1369-60002) orInterface board (LAN) (G1369C or G1369-60012)

Agilent Technologies Fast Ethernet, Ethernet/802.3, RJ-45 (10/100Base-TX) recommended for re-ordering

LAN Communication Interface board (G1369A or G1369-60001)

Agilent Technologies Fast Ethernet, Ethernet/802.3, RJ-45 (10/100Base-TX) (obsolete)

J4106A1 Hewlett Packard Ethernet/802.3, RJ-45 (10Base-T)


12 Hardware InformationOptional Interface Boards

Recommended LAN Cables

J4105A1 Hewlett Packard Token Ring/802.5, DB9, RJ-45 (10Base-T)

J4100A1 Hewlett Packard Fast Ethernet, Ethernet/802.3, RJ-45 (10/100Base-TX) + BNC (10Base2)

1 These cards may be no longer orderable. Minimum firmware of these Hewlett Packard JetDirect cards is A.05.05.

p/n Description



Table 33 LAN Boards

Type Vendor Supported networks


Hardware Information 12Electrical Connections

Electrical Connections

• The CAN bus is a serial bus with high speed data transfer. The two connectors for the CAN bus are used for internal module data transfer and synchronization.

• Two independent analog outputs provide signals for integrators or data handling.

• The interface board slot is used for external contacts and BCD bottle number output or LAN connections.

• The REMOTE connector may be used in combination with other analytical instruments from Agilent Technologies if you want to use features such as start, stop, common shut down, prepare, and so on.

• With the appropriate software, the RS- 232C connector may be used to control the module from a computer through a RS- 232C connection. This connector is activated and can be configured with the configuration switch.

• The power input socket accepts a line voltage of 100 – 240 VAC ± 10 % with a line frequency of 50 or 60 Hz. Maximum power consumption varies by module. There is no voltage selector on your module because the power supply has wide- ranging capability. There are no externally accessible fuses, because automatic electronic fuses are implemented in the power supply.

NOTE Never use cables other than the ones supplied by Agilent Technologies to ensure proper functionality and compliance with safety or EMC regulations.


12 Hardware InformationElectrical Connections

Rear view of the module

Figure 80 Rear View of Detector – Electrical Connections and Label

NOTE The GPIB interface has been removed with the introduction of the 1260 Infinity modules.


Hardware Information 12Electrical Connections

Serial Number Information

Serial Number Information 1260 Infinity

The serial number information on the instrument labels provide the following information:

Serial Number Information 1200 Series and 1290 Infinity

The serial number information on the instrument labels provide the following information:

CCXZZ00000 Format

CC Country of manufacturing• DE = Germany• JP = Japan • CN = China

X Alphabetic character A-Z (used by manufacturing)

ZZ Alpha-numeric code 0-9, A-Z, where each combination unambiguously denotes a module (there can be more than one code for the same module)

00000 Serial number

CCYWWSSSSS Format

CC country of manufacturing • DE = Germany• JP = Japan• CN = China

YWW year and week of last major manufacturing change, e.g. 820 could be week 20 of 1998 or 2008

SSSSS real serial number


12 Hardware InformationInterfaces

Interfaces

The Agilent 1200 Infinity Series modules provide the following interfaces:

Table 34 Agilent 1200 Infinity Series Interfaces

Module CAN LAN/BCD(optional)

LAN(on-board)

RS-232 Analog APG Remote

Special

Pumps

G1310B Iso PumpG1311B Quat PumpG1311C Quat Pump VLG1312B Bin PumpK1312B Bin Pump Clinical Ed.G1312C Bin Pump VL1376A Cap PumpG2226A Nano PumpG5611A Bio-inert Quat Pump

2 Yes No Yes 1 Yes

G4220A/B Bin PumpG4204A Quat Pump

2 No Yes Yes No Yes CAN-DC- OUT for CAN slaves

G1361A Prep Pump 2 Yes No Yes No Yes CAN-DC- OUT for CAN slaves

Samplers

G1329B ALSG2260A Prep ALS

2 Yes No Yes No Yes THERMOSTAT for G1330B/K1330B

G1364B FC-PSG1364C FC-ASG1364D FC-μSG1367E HiP ALSK1367E HiP ALS Clinical Ed.G1377A HiP micro ALSG2258A DL ALSG5664A Bio-inert FC-ASG5667A Bio-inert Autosampler

2 Yes No Yes No Yes THERMOSTAT for G1330B/K1330BCAN-DC- OUT for CAN slaves

G4226A ALS 2 Yes No Yes No Yes


Hardware Information 12Interfaces

Detectors

G1314B VWD VLG1314C VWD VL+

2 Yes No Yes 1 Yes

G1314E/F VWDK1314F Clinical Ed.

2 No Yes Yes 1 Yes

G4212A/B DADK4212B DAD Clinical Ed.

2 No Yes Yes 1 Yes

G1315C DAD VL+G1365C MWDG1315D DAD VLG1365D MWD VL

2 No Yes Yes 2 Yes

G1321B FLDK1321B FLD Clinical Ed.G1321C FLD

2 Yes No Yes 2 Yes

G1362A RID 2 Yes No Yes 1 Yes

G4280A ELSD No No No Yes Yes Yes EXT ContactAUTOZERO

Others

G1170A Valve Drive 2 No No No No No 1

G1316A/C TCCK1316C TCC Clinical Ed.

2 No No Yes No Yes

G1322A DEGK1322A DEG Clinical Ed.

No No No No No Yes AUX

G1379B DEG No No No Yes No Yes

G4225A DEGK4225A DEG Clinical Ed.

No No No Yes No Yes



LAN(on-board)


Special



• CAN connectors as interface to other modules

• LAN connector as interface to the control software

• RS- 232C as interface to a computer

• REMOTE connector as interface to other Agilent products

• Analog output connector(s) for signal output

G4227A Flex Cube 2 No No No No No CAN-DC- OUT for CAN slaves1

G4240A CHIP CUBE 2 Yes No Yes No Yes CAN-DC- OUT for CAN slavesTHERMOSTAT for G1330A/B (NOT USED), K1330B

1 Requires a HOST module with on-board LAN (e.g. G4212A or G4220A with minimum firmware B.06.40 or C.06.40) or with ad-ditional G1369C LAN Card



LAN(on-board)


Special

NOTE The detector (DAD/MWD/FLD/VWD/RID) is the preferred access point for control via LAN. The inter-module communication is done via CAN.



Overview Interfaces

CAN

The CAN is inter- module communication interface. It is a 2- wire serial bus system supporting high speed data communication and real- time requirement.

LAN

The modules have either an interface slot for an LAN card (e.g. Agilent G1369B/C LAN Interface) or they have an on- board LAN interface (e.g. detectors G1315C/D DAD and G1365C/D MWD). This interface allows the control of the module/system via a PC with the appropriate control software. Some modules have neither on- board LAN nor an interface slot for a LAN card (e.g. G1170A Valve Drive or G4227A Flex Cube). These are hosted modules and require a Host module with firmware B.06.40 or later or with additional G1369C LAN Card.

RS-232C (Serial)

The RS- 232C connector is used to control the module from a computer through RS- 232C connection, using the appropriate software. This connector can be configured with the configuration switch module at the rear of the module. Refer to Communication Settings for RS- 232C.

The RS- 232C is designed as DCE (data communication equipment) with a 9- pin male SUB- D type connector. The pins are defined as:

NOTE If an Agilent detector (DAD/MWD/FLD/VWD/RID) is in the system, the LAN should be connected to the DAD/MWD/FLD/VWD/RID (due to higher data load). If no Agilent detector is part of the system, the LAN interface should be installed in the pump or autosampler.

NOTE There is no configuration possible on main boards with on-board LAN. These are pre-configured for

• 19200 baud,

• 8 data bit with no parity and

• one start bit and one stop bit are always used (not selectable).



Figure 81 RS-232 Cable

Analog Signal Output

The analog signal output can be distributed to a recording device. For details refer to the description of the module’s main board.

Table 35 RS-232C Connection Table

Pin Direction Function

1 In DCD

2 In RxD

3 Out TxD

4 Out DTR

5 Ground

6 In DSR

7 Out RTS

8 In CTS

9 In RI



APG Remote

The APG Remote connector may be used in combination with other analytical instruments from Agilent Technologies if you want to use features as common shut down, prepare, and so on.

Remote control allows easy connection between single instruments or systems to ensure coordinated analysis with simple coupling requirements.

The subminiature D connector is used. The module provides one remote connector which is inputs/outputs (wired- or technique).

To provide maximum safety within a distributed analysis system, one line is dedicated to SHUT DOWN the system’s critical parts in case any module detects a serious problem. To detect whether all participating modules are switched on or properly powered, one line is defined to summarize the POWER ON state of all connected modules. Control of analysis is maintained by signal readiness READY for next analysis, followed by START of run and optional STOP of run triggered on the respective lines. In addition PREPARE and START REQUEST may be issued. The signal levels are defined as:

• standard TTL levels (0 V is logic true, + 5.0 V is false),

• fan- out is 10 ,

• input load is 2.2 kOhm against + 5.0 V, and

• output are open collector type, inputs/outputs (wired- or technique).

NOTE All common TTL circuits operate with a 5 V power supply. A TTL signal is defined as "low" or L when between 0 V and 0.8 V and "high" or H when between 2.0 V and 5.0 V (with respect to the ground terminal).



Special Interfaces

There is no special interface for this module.

Table 36 Remote Signal Distribution

Pin Signal Description

1 DGND Digital ground

2 PREPARE (L) Request to prepare for analysis (for example, calibration, detector lamp on). Receiver is any module performing pre-analysis activities.

3 START (L) Request to start run / timetable. Receiver is any module performing run-time controlled activities.

4 SHUT DOWN (L) System has serious problem (for example, leak: stops pump). Receiver is any module capable to reduce safety risk.

5 Not used

6 POWER ON (H) All modules connected to system are switched on. Receiver is any module relying on operation of others.

7 READY (H) System is ready for next analysis. Receiver is any sequence controller.

8 STOP (L) Request to reach system ready state as soon as possible (for example, stop run, abort or finish and stop injection). Receiver is any module performing run-time controlled activities.

9 START REQUEST (L) Request to start injection cycle (for example, by start key on any module). Receiver is the autosampler.


Hardware Information 12Setting the 8-bit Configuration Switch (without On-board LAN)

Setting the 8-bit Configuration Switch (without On-board LAN)

The 8- bit configuration switch is located at the rear of the module.

This module does not have its own on- board LAN interface. It can be controlled through the LAN interface of another module, and a CAN connection to that module.

Figure 82 Configuration switch (settings depend on configured mode)

All modules without on- board LAN:

• default should be ALL DIPS DOWN (= best settings)

• Bootp mode for LAN and

• 19200 baud, 8 data bit / 1 stop bit with no parity for RS- 232

• DIP 1 DOWN and DIP 2 UP allows special RS- 232 settings

• for boot/test modes DIPS 1+2 must be UP plus required mode

Switch settings provide configuration parameters for serial communication protocol and instrument specific initialization procedures.

NOTE For normal operation use the default (best) settings.

NOTE With the introduction of the Agilent 1260 Infinity, all GPIB interfaces have been removed. The preferred communication is LAN.


12 Hardware InformationSetting the 8-bit Configuration Switch (without On-board LAN)

Communication Settings for RS-232C

The communication protocol used in the column compartment supports only hardware handshake (CTS/RTR).

Switches 1 in down and 2 in up position define that the RS- 232C parameters will be changed. Once the change has been completed, the column instrument must be powered up again in order to store the values in the non- volatile memory.

Use the following tables for selecting the setting which you want to use for RS- 232C communication. The number 0 means that the switch is down and 1 means that the switch is up.

NOTE The following tables represent the configuration switch settings for the modules without on-board LAN only.

Table 37 8-bit Configuration Switch (without on-board LAN)

Mode Select 1 2 3 4 5 6 7 8

RS-232C 0 1 Baudrate Data Bits

Parity

Reserved 1 0 Reserved

TEST/BOOT 1 1 RSVD SYS RSVD RSVD FC

NOTE The LAN settings are done on the LAN Interface Card G1369B/C. Refer to the documentation provided with the card.

Table 38 Communication Settings for RS-232C Communication (without on-board LAN)

Mode Select

1 2 3 4 5 6 7 8

RS-232C 0 1 Baudrate Data Bits Parity


Hardware Information 12Setting the 8-bit Configuration Switch (without On-board LAN)

One start bit and one stop bit are always used (not selectable).

Per default, the module will turn into 19200 baud, 8 data bit with no parity.

Table 39 Baudrate Settings (without on-board LAN)

Switches Baud Rate Switches Baud Rate

3 4 5 3 4 5

0 0 0 9600 1 0 0 9600

0 0 1 1200 1 0 1 14400

0 1 0 2400 1 1 0 19200

0 1 1 4800 1 1 1 38400

Table 40 Data Bit Settings (without on-board LAN)

Switch 6 Data Word Size

0 7 Bit Communication

1 8 Bit Communication

Table 41 Parity Settings (without on-board LAN)

Switches Parity

7 8

0 0 No Parity

0 1 Odd Parity

1 1 Even Parity


12 Hardware InformationSetting the 8-bit Configuration Switch (without On-board LAN)

Special Settings

The special settings are required for specific actions (normally in a service case).

Boot-Resident

Firmware update procedures may require this mode in case of firmware loading errors (main firmware part).

If you use the following switch settings and power the instrument up again, the instrument firmware stays in the resident mode. It is not operable as a module. It only uses basic functions of the operating system for example, for communication. In this mode the main firmware can be loaded (using update utilities).

Forced Cold Start

A forced cold start can be used to bring the module into a defined mode with default parameter settings.

If you use the following switch settings and power the instrument up again, a forced cold start has been completed.

Table 42 Boot Resident Settings (without on-board LAN)

Mode Select SW1 SW2 SW3 SW4 SW5 SW6 SW7 SW8

TEST/BOOT 1 1 0 0 1 0 0 0

CAUTION Loss of data

Forced cold start erases all methods and data stored in the non-volatile memory. Exceptions are calibration settings, diagnosis and repair log books which will not be erased.

➔ Save your methods and data before executing a forced cold start.

Table 43 Forced Cold Start Settings (without on-board LAN)

Mode Select SW1 SW2 SW3 SW4 SW5 SW6 SW7 SW8

TEST/BOOT 1 1 0 0 0 0 0 1


Hardware Information 12Early Maintenance Feedback

Early Maintenance Feedback

Maintenance requires the exchange of components which are subject to wear or stress. Ideally, the frequency at which components are exchanged should be based on the intensity of usage of the module and the analytical conditions, and not on a predefined time interval. The early maintenance feedback (EMF) feature monitors the usage of specific components in the instrument, and provides feedback when the user- selectable limits have been exceeded. The visual feedback in the user interface provides an indication that maintenance procedures should be scheduled.

EMF Counters

EMF counters increment with use and can be assigned a maximum limit which provides visual feedback in the user interface when the limit is exceeded. Some counters can be reset to zero after the required maintenance procedure.

Using the EMF Counters

The user- settable EMF limits for the EMF Counters enable the early maintenance feedback to be adapted to specific user requirements. The useful maintenance cycle is dependent on the requirements for use. Therefore, the definition of the maximum limits need to be determined based on the specific operating conditions of the instrument.

Setting the EMF Limits

The setting of the EMF limits must be optimized over one or two maintenance cycles. Initially the default EMF limits should be set. When instrument performance indicates maintenance is necessary, take note of the values displayed by the EMF counters. Enter these values (or values slightly less than the displayed values) as EMF limits, and then reset the EMF counters to zero. The next time the EMF counters exceed the new EMF limits, the EMF flag will be displayed, providing a reminder that maintenance needs to be scheduled.


12 Hardware InformationInstrument Layout

Instrument Layout

The industrial design of the module incorporates several innovative features. It uses Agilent’s E- PAC concept for the packaging of electronics and mechanical assemblies. This concept is based upon the use of expanded polypropylene (EPP) layers of foam plastic spacers in which the mechanical and electronic boards components of the module are placed. This pack is then housed in a metal inner cabinet which is enclosed by a plastic external cabinet. The advantages of this packaging technology are:

• virtual elimination of fixing screws, bolts or ties, reducing the number of components and increasing the speed of assembly/disassembly,

• the plastic layers have air channels molded into them so that cooling air can be guided exactly to the required locations,

• the plastic layers help cushion the electronic and mechanical parts from physical shock, and

• the metal inner cabinet shields the internal electronics from electromagnetic interference and also helps to reduce or eliminate radio frequency emissions from the instrument itself.



13Appendix

General Safety Information 246

The Waste Electrical and Electronic Equipment (WEEE) Directive (2002/96/EC) 249

Lithium Batteries Information 250

Radio Interference 251

Sound Emission 252

UV-Radiation (UV-lamps only) 253

Solvent Information 254

Installation of Stainless Steel Cladded PEEK Capillaries 256

First Step: Finger-tight Fitting 257

Second Step: Installation to Connector 257

Removing Capillaries 261

Agilent Technologies on Internet 262

This chapter provides safetey and other general information.


13 AppendixGeneral Safety Information

General Safety Information

Safety Symbols

Table 44 Safety Symbols

Symbol Description

The apparatus is marked with this symbol when the user should refer to the instruction manual in order to protect risk of harm to the operator and to protect the apparatus against damage.

Indicates dangerous voltages.

Indicates a protected ground terminal.

Indicates eye damage may result from directly viewing the light produced by the deuterium lamp used in this product.

The apparatus is marked with this symbol when hot surfaces are available and the user should not touch it when heated up.

WARNING A WARNING

alerts you to situations that could cause physical injury or death.

➔ Do not proceed beyond a warning until you have fully understood and met the indicated conditions.

CAUTION A CAUTION

alerts you to situations that could cause loss of data, or damage of equipment.

➔ Do not proceed beyond a caution until you have fully understood and met the indicated conditions.


Appendix 13General Safety Information

General Safety Information

The following general safety precautions must be observed during all phases of operation, service, and repair of this instrument. Failure to comply with these precautions or with specific warnings elsewhere in this manual violates safety standards of design, manufacture, and intended use of the instrument. Agilent Technologies assumes no liability for the customer’s failure to comply with these requirements.

Safety Standards

This is a Safety Class I instrument (provided with terminal for protective earthing) and has been manufactured and tested according to international safety standards.

Operation

Before applying power, comply with the installation section. Additionally the following must be observed.

Do not remove instrument covers when operating. Before the instrument is switched on, all protective earth terminals, extension cords, auto- transformers, and devices connected to it must be connected to a protective earth via a ground socket. Any interruption of the protective earth grounding will cause a potential shock hazard that could result in serious personal injury. Whenever it is likely that the protection has been impaired, the instrument must be made inoperative and be secured against any intended operation.

Make sure that only fuses with the required rated current and of the specified type (normal blow, time delay, and so on) are used for

WARNING Ensure the proper usage of the equipment.

The protection provided by the equipment may be impaired.

➔ The operator of this instrument is advised to use the equipment in a manner as specified in this manual.


13 AppendixGeneral Safety Information

replacement. The use of repaired fuses and the short- circuiting of fuse holders must be avoided.

Some adjustments described in the manual, are made with power supplied to the instrument, and protective covers removed. Energy available at many points may, if contacted, result in personal injury.

Any adjustment, maintenance, and repair of the opened instrument under voltage should be avoided whenever possible. When inevitable, this has to be carried out by a skilled person who is aware of the hazard involved. Do not attempt internal service or adjustment unless another person, capable of rendering first aid and resuscitation, is present. Do not replace components with power cable connected.

Do not operate the instrument in the presence of flammable gases or fumes. Operation of any electrical instrument in such an environment constitutes a definite safety hazard.

Do not install substitute parts or make any unauthorized modification to the instrument.

Capacitors inside the instrument may still be charged, even though the instrument has been disconnected from its source of supply. Dangerous voltages, capable of causing serious personal injury, are present in this instrument. Use extreme caution when handling, testing and adjusting.

When working with solvents, observe appropriate safety procedures (for example, goggles, safety gloves and protective clothing) as described in the material handling and safety data sheet by the solvent vendor, especially when toxic or hazardous solvents are used.


Appendix 13The Waste Electrical and Electronic Equipment (WEEE) Directive (2002/96/EC)

The Waste Electrical and Electronic Equipment (WEEE) Directive (2002/96/EC)

Abstract

The Waste Electrical and Electronic Equipment (WEEE) Directive (2002/96/EC), adopted by EU Commission on 13 February 2003, is introducing producer responsibility on all Electric and Electronic appliances from 13 August 2005.

NOTE

This product complies with the WEEE Directive (2002/96/EC) marking requirements. The affixed label indicates that you must not discard this electrical/electronic product in domestic household waste.

Product Category: With reference to the equipment types in the WEEE Directive Annex I, this product is classed as a “Monitoring and Control instrumentation” product.

Do not dispose off in domestic household waste

To return unwanted products, contact your local Agilent office, or see www.agilent.com for more information.


13 AppendixLithium Batteries Information

Lithium Batteries Information

WARNING Lithium batteries may not be disposed-off into the domestic waste. Transportation of discharged Lithium batteries through carriers regulated by IATA/ICAO, ADR, RID, IMDG is not allowed.

Danger of explosion if battery is incorrectly replaced.

➔ Discharged Lithium batteries shall be disposed off locally according to national waste disposal regulations for batteries.

➔ Replace only with the same or equivalent type recommended by the equipment manufacturer.

WARNING Lithiumbatteri - Eksplosionsfare ved fejlagtig håndtering.

Udskiftning må kun ske med batteri af samme fabrikat og type.

➔ Lever det brugte batteri tilbage til leverandøren.

WARNING Lithiumbatteri - Eksplosionsfare.

Ved udskiftning benyttes kun batteri som anbefalt av apparatfabrikanten.

➔ Brukt batteri returneres appararleverandoren.

NOTE Bij dit apparaat zijn batterijen geleverd. Wanneer deze leeg zijn, moet u ze niet weggooien maar inleveren als KCA.


Appendix 13Radio Interference

Radio Interference

Cables supplied by Agilent Technologies are screened to provide optimized protection against radio interference. All cables are in compliance with safety or EMC regulations.

Test and Measurement

If test and measurement equipment is operated with unscreened cables, or used for measurements on open set- ups, the user has to assure that under operating conditions the radio interference limits are still met within the premises.


13 AppendixSound Emission

Sound Emission

Manufacturer’s Declaration

This statement is provided to comply with the requirements of the German Sound Emission Directive of 18 January 1991.

This product has a sound pressure emission (at the operator position) < 70 dB.

• Sound Pressure Lp < 70 dB (A)

• At Operator Position

• Normal Operation

• According to ISO 7779:1988/EN 27779/1991 (Type Test)


Appendix 13UV-Radiation (UV-lamps only)

UV-Radiation (UV-lamps only)

Emissions of ultraviolet radiation (200- 315 nm) from this product is limited such that radiant exposure incident upon the unprotected skin or eye of operator or service personnel is limited to the following TLVs (Threshold Limit Values) according to the American Conference of Governmental Industrial Hygienists:

Typically the radiation values are much smaller than these limits:

Table 45 UV-Radiation Limits

Exposure/day Effective Irradiance

8 hours 0.1 µW/cm2

10 minutes 5.0 µW/cm2

Table 46 UV-Radiation Typical Values

Position Effective Irradiance

Lamp installed, 50-cm distance average 0.016 µW/cm2

Lamp installed, 50-cm distance maximum 0.14 µW/cm2


13 AppendixSolvent Information

Solvent Information

Flow Cell

To protect optimal functionality of your flow- cell:

• Avoid the use of alkaline solutions (pH > 9.5) which can attack quartz and thus impair the optical properties of the flow cell.

• If the flow cell is transported while temperatures are below 5 °C, it must be assured that the cell is filled with alcohol.

• Aqueous solvents in the flow cell can built up algae. Therefore do not leave aqueous solvents sitting in the flow cell. Add a small % of organic solvents (e.g. acetonitrile or methanol ~5 %).

Use of Solvents

Observe the following recommendations on the use of solvents.

• Brown glass ware can avoid growth of algae.

• Small particles can permanently block capillaries and valves. Therefore always filter solvents through 0.4 µm filters.

• Avoid the use of the following steel- corrosive solvents:

• Solutions of alkali halides and their respective acids (for example, lithium iodide, potassium chloride, and so on),

• High concentrations of inorganic acids like sulfuric acid and nitric acid, especially at higher temperatures (if your chromatography method allows, replace by phosphoric acid or phosphate buffer which are less corrosive against stainless steel),

• Halogenated solvents or mixtures which form radicals and/or acids, for example:

2CHCl3 + O2→ 2COCl2 + 2HCl

This reaction, in which stainless steel probably acts as a catalyst, occurs quickly with dried chloroform if the drying process removes the stabilizing alcohol,


Appendix 13Solvent Information

• Chromatographic grade ethers, which can contain peroxides (for example, THF, dioxane, di- isopropylether) such ethers should be filtered through dry aluminium oxide which adsorbs the peroxides,

• Solvents containing strong complexing agents (e.g. EDTA),

• Mixtures of carbon tetrachloride with 2- propanol or THF.


13 AppendixInstallation of Stainless Steel Cladded PEEK Capillaries

Installation of Stainless Steel Cladded PEEK Capillaries

The 1260 Infinity Bio- inert LC system uses PEEK capillaries that are cladded with stainless steel. These capillaries combine the high pressure stability of steel with the inertness of PEEK. They are used in the high pressure flow path after sample introduction (loop/needle seat capillary) through the thermostatted column compartment/heat exchangers to the column. Such capillaries need to be installed carefully in order to keep them tight without damaging them by over- tightening.

Installation procedure

The amount of force/torque needing to be applied to install the capillary depends on

• the female connector to which the capillary is installed, and whether the material of that connector is soft or hard. Compared with hard connectors, a greater tightening angle is required for soft connectors to achieve same torque.

• whether the capillary is installed for the first time or subsequent times. For the first time, a greater tightening angle needs to be applied.

The installation consists of two steps. In the first step, the fitting is installed finger- tight without using tools. Finger- tight means that the fitting will grip and hold the capillary. This brings the fitting to the appropriate start position (marked as 0 ° below) for the second step.

NOTE This installation procedure applies for capillaries and corresponding fittings used in modules delivered before January 2013. For current capillaries and fittings, see “Installing UHP-FF Fittings” on page 69.

CAUTION Handling of stainless-steel-cladded PEEK capillaries

Be careful when installing stainless-steel-cladded PEEK capillaries. The correct torque must be applied to avoid leaks potentially causing measurement problems or damage to the capillary.

➔ Follow the procedure below for a correct installation


Appendix 13Installation of Stainless Steel Cladded PEEK Capillaries

First Step: Finger-tight Fitting

1 Tighten the fitting using your fingers.

Second Step: Installation to Connector

In the second step (“Second Step: Installation to Hard Connectors” on page 257 or “Second Step: Installation to Soft Connectors” on page 258), a wrench is used to rotate the fitting relative to the finger- tight position by a defined angle. For each of the cases mentioned above, there is a recommended range in which the fitting is tight.

Staying below this range could create a leak, either a visible one or a micro- leak, potentially biasing measurement results. Exceeding the recommended range could damage the capillary.

Alternatively, a torque wrench may be used. The target torque for all connections is about 0.7 Nm. When using a torque wrench, read instructions for that tool carefully, as wrong handling may easily miss the correct torque.

Second Step: Installation to Hard Connectors

Use this procedure for hard connectors made from metal (titanium) or ceramics. In the system, these are connections to and from the analytical head of the autosampler (connections to injection valve and needle), and to a metal column.



First installation of a capillary to a hard connector

1 When tightening a fitting for the first time, start from the finger- tight position (which is not necessarily a vertical wrench position) and rotate the wrench by 135 – 180 °. Staying below 135 ° (grey arrow) will be insufficiently tight, more than 180 ° (red arrow) could damage the capillary.

Second and subsequent installations of a capillary to a hard connector

1 When tightening the fitting for the second and subsequent times, again start from the finger- tight position (which is not necessarily a vertical wrench position) and rotate the wrench by 90 – 135 °. Staying below 90 ° (grey arrow) could be insufficiently tight, more than 135 ° (red arrow) could damage the capillary.

Second Step: Installation to Soft Connectors

Use this procedure for soft connectors, which are typically made from PEEK. These are the following connections:

• to and from all bio- inert valves (injection valve in the autosampler and valves in the thermostatted column compartment and 1290 Infinity Valve Drive),

• bio- inert ZDV unions (detector flow cells, multi- draw upgrade kit, capillary to capillary connections, for example, for heat exchangers),



• to the autosampler needle and

• to PEEK columns (like many bio- inert columns).

For the installation of bio- inert ZDV unions, see “Installation of the Bio- inert Zero Dead Volume (ZDV) Union” on page 73.

First installation of a capillary to a soft connector

1 When tightening a fitting for the first time, start from the finger- tight position (which does not necessarily need to be a vertical wrench position) and rotate the wrench by 180 – 210 °. Staying below 180 ° (grey arrow) will not be sufficiently tight, more than 210 ° (red arrow) could damage the capillary.

Second and subsequent installations of a capillary to a soft connector

1 When tightening the fitting for the second and subsequent times, again start from the finger- tight position (which is not necessarily a vertical wrench position) and rotate the wrench by 135 – 180 °. Staying below 135 ° (grey arrow) could be insufficiently tight enough, more than 180 ° (red arrow) could damage the capillary.



Summary for Second Step

Table 47 Summary for second step

2ndStep First installation Subsequent installations

Hard connectors

Soft connectors



Removing Capillaries

To keep the flow path free of stainless steel, the front end of the capillary is made of PEEK. Under high pressure, or when in contact with some solvents, PEEK can expand to the shape of the connector where the capillary is installed. If the capillary is removed, this may become visible as a small step. In such cases, do not try to pull the fitting from the capillary, as this can destroy the front part of the capillary. Instead, carefully pull it to the rear. During installation of the capillary, the fitting will end up in the correct position.

Figure 83 Capillary fitting

CAUTION Potential damage of capillaries

➔ Do not remove fittings from used capillaries.


13 AppendixAgilent Technologies on Internet

Agilent Technologies on Internet

For the latest information on products and services visit our worldwide web site on the Internet at:

http://www.agilent.com


Index

Index

88-bit configuration switch

without On-Board LAN 239

Aaccessory kit parts 204

accuracy of wavelength 40

Agilent Lab Advisor software 142

Agilent Lab Advisor 142

Agilenton internet 262

algae 254, 254

algea 194

ambient non-operating temperature 39

ambient operating temperature 39

analog signal 236

analogcable 210

apg remote 237

Bbattery

safety information 250

BCD boardexternal contacts 225

BCDcable 215

bench space 38

bio-inert 189materials 32

boardHP JetDirect card 227

boards

LAN card 227

Ccable

analog 210BCD 215CAN 217connecting APG remote 56connecting CAN 56connecting LAN 56connecting the ChemStation 56connecting the power 56external contact 218LAN 217remote 212RS-232 219

cablesanalog 208BCD 208CAN 209LAN 209overview 208remote 208RS-232 209

calibration sample 177

CANcable 217

cautions and warnings 185

cleaning 188

Communication settingsRS-232C 240

compensation sensor open 149

compensation sensor short 150

condensation 38

configuration

one stack 53two stack 55

cut-off filter 16

cuvettehow to use 193

Ddefect on arrival 50

degradation UV 18, 160

delivery checklist 51

dimensions 39

Eelectrical connections

descriptions of 229

EMFearly maintenance feedback 243

emission condenser lens 16

emission grating 16

emission monochromator 20

emission slit 16

error messagesA/D Overflow 152ADC Not Calibrated 152compensation sensor open 149compensation sensor short 150fan failed 150Flash Lamp Current Overflow 153Flash Trigger Lost 154FLF Board not found 151Flow Cell Removed 155Lamp Cover Open 151leak sensor open 148leak sensor short 149


Index

leak 148lost CAN partner 147motor errors 156remote timeout 147shutdown 146timeout 146Wavelength Calibration Failed 154Wavelength Calibration Lost 155

excitation condenser lens 16

excitation grating 16

excitation monochromator 18

excitation slit 16

external contactcable 218

external contactsBCD board 225

Ffan failed 150

featuressafety and maintenance 42, 44, 48

firmwaredescription 222main system 222resident system 222update tool 223updates 222, 198upgrade/downgrade 198

flow cell 16, 20, 254solvent information 254

fluorescence and phosphorescence 13

fluorescence detection 24

frequency range 39

front view of module 61

Ggeneral error messages 146

GLP features 42, 45, 48

glycogen 177

HHow the Detector Operates 12

how to use the cuvette 193

HP JetDirect card 227

humidity 39

Iinstallation

bench space 38flow connections 64of flow cell and capillaries 64of the detector 61site requirements 35

instrument layout 244

interfaces 232

internet 262

Introduction to the Detector 10

Llamp intensity history 161

lamp intensity test 160

LANcable 217communication interface board 227

leak sensor open 148

leak sensor short 149

leak 148

leakscorrecting 195

line frequency 39

line voltage 39

lithium batteries 250

lost CAN partner 147

luminescence 12

Mmaintenance

definition of 184

feedback 243overview 187replacing firmware 198

materialsbio-inert 32

messageA/D Overflow 152ADC Not Calibrated 152Flash Lamp Current Overflow 153Flash Trigger Lost 154FLF Board not found 151Flow Cell Removed 155Lamp Cover Open 151motor messages 156remote timeout 147Wavelength Calibration Failed 154Wavelength Calibration Lost 155

method development1 - check the LC system for impurities 832 - optimize limits of detection and selectivity 843 - set up routine methods 95multi wavelength detection 95take a fluorescence scan 85

mirror 16

monochromatorEM 20, 16EX 18, 16

multi wavelength detection 95

Nnon-operating altitude 39

non-operating temperature 39

Ooff-line measurements 10

operating Altitude 39

operating temperature 39

operation of the detector 12


Index

optical unit overview 16

optimizationexample 99stack configuration 52

Ppackaging

damaged 50

parts identificationaccessory kit 204cables 207overview 202

partsdamaged 51missing 51

peakwidth settings 134

peakwidthselecting 133

performance specifications 40, 43, 46

phosphorescence detection 25

photoluminescence 12

photo-multiplier tubelocation of PMT 16PMT 21

physical specifications 39

PMTgain test 120gain 125, 21photo-multiplier tube 21range 27

power consideration 36

power consumption 39

power cords 37

power supply indicator 139

Rradio interference 251

Raman S/N test 162

Raman 15

recalibration of wavelength 138, 159

reference diode 23

reference system 23, 23

remotecable 212

repairscautions and warnings 185correction leaks 195exchanging a flow cell 189of the detector 183replacing firmware 198replacing leak handling system 196

replacing interface board (BCD/LAN) 197

response time settings 134

response timeselecting 133

responsetime 27

RS-232Ccable 219communication settings 240

Ssafety class I 247

safety informationlithium batteries 250

safetygeneral information 247standards 39symbols 246

selectingpeakwidth 133response time 133

serial numberinformation 231, 231

settingspeakwidth 134response time 134

shutdown 146

site requirements 35power cords 37

solvent information 254

solvents 254

sound emission 252

special interfaces 238

special settingsboot-resident 242forced cold start 242

specificationphysical 39

specificationsanalog outputs 42, 44, 47communications 42, 44, 47flow cell 41, 44, 47GLP features 42, 45, 48monochromators 40, 43, 46performance 40, 43, 46pulse frequency 40, 43, 46safety and maintenance 42, 44, 48wavelength accuracy 40

spectra wavelength shift 121

stack configurationfront view 55rear view 56

status indicator 140

stray light 135

system setup and installationoptimizing stack configuration 52

Ttemperature sensor 148

test chromatogram 166

test functions 138, 159

testsfunctions 159lamp intensity history 161lamp intensity 160PMT gain test 120


Index

Raman AST S/N 162test chromatogram 166

timeout 146

troubleshootingerror messages 138, 145status indicators 139, 138

Uunpacking 50

UV degradation 18, 160

Vvoltage range 39

Wwarnings and cautions 185

wavelength calibration procedure 168, 177

wavelength calibration 168

wavelength shift of spectra 121

wavelengthrecalibration 138, 159

weight 39

Xxenon flash lamp 16, 17


Index


www.agilent.com

In This Book

This manual contains technical reference information about the Agilent 1260 Infinity Fluorescence Detector (G1321B SPECTRA, G1321C),and Agilent 1100/1200 Series Fluorescence Detector G1321A (obsolete).

• introduction and specifications,

• installation,

• using and optimizing,

• troubleshooting and diagnose,

• maintenance,

• parts identification,

• safety and related information.

© Agilent Technologies 2010-2012, 2013

Printed in Germany 11/2013

*G1321-90014**G1321-90014*G1321- 90014Rev. B

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